Nucleic acid and corresponding protein entitled 85P1B3 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 85P1B3) and its encoded protein are described. While 85P1B3 exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed in multiple cancers including set forth in Table 1. Consequently, 85P1B3 provides a diagnostic and/or therapeutic target for cancers. The 85P1B3 gene or fragment thereof, or its encoded protein or a fragment thereof, can be used to elicit an immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. patentapplication Ser. No. 09/942,052, filed Aug. 28, 2001, now U.S. Pat. No.______, which claims the benefit of U.S. provisional patent applicationSer. No. 60/228,432, filed Aug. 28, 2000, the entire contents of whichare hereby incorporated herein by reference.

SUBMISSION ON COMPACT DISC

The content of the following submission on compact discs is incorporatedherein by reference in its entirety: A compact disc copy of the SequenceListing (COPY 1) (file name: 5115820028.01, date recorded: Jul. 17,2006, size: 198,656 bytes); a duplicate compact disc copy of theSequence Listing (COPY 2) (file name: 5115820028.01, date recorded: Jul.17, 2006, size: 198,656 bytes); and a computer readable form copy of theSequence Listing (CRF COPY) (file name: 5115820028.01, date recorded:Jul. 17, 2006, size: 198,656 bytes).

TECHNICAL FIELD

The invention described herein relates to a novel gene and its encodedprotein, termed 85P1B3, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express85P1B3.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein, et al., Nat. Med. (1997)3:402). More recently identified prostate cancer markers include PCTA-1(Su, et al., Proc. Natl. Acad. Sci. USA (1996) 93:7252),prostate-specific membrane (PSM) antigen (Pinto, et al., Clin Cancer Res(1996) 2:1445-1451), STEAP (Hubert, et al., Proc. Natl. Acad. Sci. USA(1999) 96:14523-14528) and prostate stem cell antigen (PSCA) (Reiter, etal., Proc. Natl. Acad. Sci. USA (1998) 95:1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980's, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

DISCLOSURE OF THE INVENTION

The present invention relates to a novel gene, designated 85P1B3, thatis over-expressed in multiple cancers listed in Table I. Northern blotexpression analysis of 85P1B3 gene expression in normal tissues shows arestricted expression pattern in adult tissues. The nucleotide (FIG. 2)and amino acid (FIG. 2, and FIG. 3) sequences of 85P1B3 are provided.The tissue-related profile of 85P1B3 in normal adult tissues, combinedwith the over-expression observed in prostate and other tumors, showsthat 85P1B3 is aberrantly over-expressed in at least some cancers, andthus serves as a useful diagnostic and/or therapeutic target for cancersof the tissues such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 85P1B3 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding85P1B3-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a85P1B3-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the85P1B3 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 85P1B3 genes, mRNAs, or to85P1B3-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 85P1B3. Recombinant DNA moleculescontaining 85P1B3 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 85P1B3gene products are also provided. The invention further providesantibodies that bind to 85P1B3 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker.

The invention further provides methods for detecting the presence andstatus of 85P1B3 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 85P1B3. Atypical embodiment of this invention provides methods for monitoring85P1B3 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 85P1B3such as prostate cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 85P1B3 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. 85P1B3 SSH sequence. The 85P1B3 SSH sequence contains 259 bp.(SEQ ID NO: 724) and it's alignment with a fragment of the Homo sapiensOpa-interacting protein OIP5 (SEQ ID NO: 725) cDNA.

FIG. 2. The cDNA (SEQ ID NO: 727) and amino acid sequence (SEQ ID NO:728) of 85P1B3. The start methionine is underlined. The open readingframe extends from nucleic acid 13 to 702 including the stop codon.

FIG. 3. Amino acid sequence of 85P1B3 (SEQ ID. NO.:728). The 85P1B3protein has 229 amino acids.

FIG. 4. Sequence alignment of 85P1B3 (SEQ ID NO: 728) with GenBankaccession number AAC39561.1 (AF025441), Opa-interacting protein OIP5(SEQ ID NO: 731).

FIG. 5. Hydrophilicity amino acid profile of 85P1B3 determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp, T. P., et al., Proc. Natl. Acad. Sci. USA (1981) 78:3824-3828)accessed on the Protscale website through the ExPasy molecular biologyserver.

FIG. 6. Hydropathicity amino acid profile of 85P1B3 determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte, J., et al., J. Mol. Biol. (1982) 157:105-132) accessedon the ProtScale website through the ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of 85P1B3determined by computer algorithm sequence analysis using the method ofJanin (Janin, J., Nature (1979) 277:491-492) accessed on the ProtScalewebsite through the ExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of 85P1B3 determined bycomputer algorithm sequence analysis using the method of Bhaskaran andPonnuswamy (Bhaskaran, R., et al., Int. J. Pept. Protein Res. (1988)32:242-255) accessed on the ProtScale website through the ExPasymolecular biology server.

FIG. 9. Beta-turn amino acid profile of 85P1B3 determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., et al., Protein Engineering (1987) 1:289-294) accessed onthe ProtScale website through the ExPasy molecular biology server.

FIG. 10. RT-PCR analysis of 85P1B3 expression. First strand cDNA wasprepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2(VP2, pancreas, spleen and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, bladder cancerpool, kidney cancer pool, colon cancer pool, lung cancer pool, ovarycancer pool, breast cancer pool, and cancer metastasis pool.Normalization was performed by PCR using primers to actin and GAPDH.Semi-quantitative PCR, using primers to 85P1B3, was performed at 26 and30 cycles of amplification.

FIG. 11. Expression of 85P1B3 in normal human tissues. Two multipletissue northern blots (Clontech) with 2 μg of mRNA/lane, were probedwith the 85P1B3 SSH sequence. Size standards in kilobases (kb) areindicated on the side. The results show exclusive expression of anapproximately 1.4 kb 85P1B3 transcript in testis but not in any othernormal tissues.

FIG. 12. Expression of 85P1B3 in human cancer cell lines. RNA wasextracted from a panel of human cancer cell lines. Northern blots with10 μg of total RNA/lane were probed with the 85P1B3 SSH sequence. Sizestandards in kilobases (kb) are indicated on the side.

FIG. 13. Expression of 85P1B3 in human patient cancer specimens andcancer cell lines. Expression of 85P1B3 was assayed in a panel of humancancers (T) and their respective matched normal tissues (N) on RNA dotblots. 85P1B3 expression was detected in the cancers of the breast,prostate, uterus, cervix, stomach and lung. 85P1B3 was also found to behighly expressed in all human cancer cell lines tested.

FIG. 14. Expression of 85P1B3 in colon cancer patient specimens. RNA wasextracted from colon cancer cell lines (CL), normal colon (N), colontumors (T) and their normal adjacent tissues (Nat) derived from coloncancer patients. Northern blots with 10 μg of total RNA/lane were probedwith the 85P1B3 SSH sequence. Size standards in kilobases (kb) areindicated on the side. Results show expression of 85P1B3 in 2 colontumor specimens but not in the corresponding normal adjacent tissue.Expression is also seen in all 4 colon cancer cell lines (Colo 205,LoVo, T84, Caco-2). P1—Stage III, T2N1MX; P2—Stage III, T3N1MX.

FIG. 15. Expression of 85P1B3 in bladder cancer patient specimens. RNAwas extracted from bladder cancer cell lines (CL), normal bladder (N),bladder tumors (T) and their normal adjacent tissue (Nat) derived frombladder cancer patients. Northern blot with 10 μg of total RNA/lane wereprobed with the 85P1B3 SSH sequence. Size standards in kilobases (kb)are indicated on the side. Results show expression of 85P1B3 in 3 of 5bladder tumor specimens. Expression is also seen in all three bladdercancer cell lines, UM-UC-3, J82, and SCABER.

FIG. 16. Expression of 85P1B3 in lung cancer patient specimens. RNA wasextracted from lung cancer cell lines (CL), normal lung (N), lung tumors(T) and their normal adjacent tissue (NAT) derived from lung cancerpatients. Northern blot with 10 μg of total RNA/lane was probed with the85P1B3 SSH sequence. Size standards in kilobases (kb) are indicated onthe side. Results show expression of 85P1B3 in three lung tumorspecimens. Expression is also seen in all lung cancer cell lines.

FIG. 17. Expression of 85P1B3 in Prostate Cancer Xenografts FollowingCastration. Male mice were injected with LAPC-9AD tumor cells. Whentumor reached a palpable size (0.3-0.5 cm in diameter), mice werecastrated and tumors harvested at different time points followingcastration. RNA was isolated from the xenograft tissues. Northern blotswith 10 μg of total RNA/lane were probed with the 85P1B3 SSH fragment.Size standards in kilobases (kb) are indicated on the side. Results showexpression of 85P1B3 is maintained following castration. A picture ofthe ethidium-bromide staining of the RNA gel is also presented.Hybridization of the same northern blot with the androgen-dependent geneTMPRSS2 confirms the quality of the androgen deprivation followingcastration.

FIG. 18. Expression of 85P1B3 in PC3 Cells Following Retroviral-MediatedGene Delivery. PC3 cells were transduced with the pSRα retroviral vectorencoding the 85P1B3 gene. Following selection with neomycin, the cellswere expanded and RNA was extracted. Northern blot with 10 μg of totalRNA/lane was probed with the 85P1B3 SSH sequence. Size standards inkilobases (kb) are indicated on the side. Results show expression of the85P1B3 transcript driven from the retroviral LTR, which migrates slowerthan the endogenous 1.4 kb 85P1B3 transcript. LAPC-9AI shows onlyexpression of the endogenous 85P1B3, but not the pSRα transcript.

FIG. 19. Schematic diagram of the alignment of 85P1B3 with its splicevariant. The region of homology between 85P1B3 and its splice variant 1is marked with a hatched box. Regions specific for 85P1B3 are marked inwhite boxes, and the ones specific for the splice variant 1 as blackboxes. The SSH sequence of 85P1B3 is also indicated by a white box.

FIG. 20. Western analysis of 85P1B3 expression with an anti-85P1B3polyclonal antibody. Panel A. Detection of GST-85P1B3 withanti-GST-85P1B3 rabbit serum. 200 ng of GST-85P1B3 (amino acids 1-229)and 200 ng of GST alone were separated by SDS-PAGE and transferred tonitrocellulose. The blot was then incubated with indicated dilutions ofanti-85P1B3 serum. Immunoreactive bands were detected by incubation withanti-rabbit IgG HRP-secondary antibody and visualized by enhancedchemiluminescence and exposure to autoradiography film. Shown witharrows is detection of the GST-85P1B3 protein and minimal detection ofGST alone. Panel B. 293T cells were transiently transfected with eitherempty pcDNA 3.1 vector or pcDNA 3.1 carrying the 85P1B3 cDNA. Lysates ofthe cells were separated by SDS-PAGE and subjected to Western analysisas performed for the data in Panel A, with 2 μg/ml of purifiedanti-85P1B3 polyclonal antibody. Panel C. Western analysis was carriedout as for the data in Panel B, but using an anti-His polyclonalantibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Arrows indicatethe immunoreactive bands corresponding to His-tagged 85P1B3 protein.

FIG. 21. Secondary structure and transmembrane prediction for 85P1B3.Panel A. The secondary structure of 85P1B3 protein was predicted usingthe HNN—Hierarchical Neural Network method (Guermeur, 1997), accessedfrom the ExPasy molecular biology server. This method indicates thepresence and location of alpha helices (h), extended strands (e), andrandom coils (c) from the primary protein sequence. The percent of theprotein in a given secondary structure is also given. Panel B. Schematicrepresentation of the probability of existence of transmembrane regionsof 85P1B3 based on the TMpred algorithm of Hofmann and Stoffel whichutilizes TMBASE (Hofmann, K., et al., Biol. Chem. Hoppe-Seyler (1993)374:166). Stretches of amino acids approximately 17-33 amino acids inlength with a value greater than 0 are potential transmembrane helices.This program indicates the presence of one helix in 85P1B3. Panel C.Schematic representation of the probability of the existence oftransmembrane regions and the extracellular and intracellularorientation of 85P1B3 based on the algorithm of Sonnhammer, von Heijne,and Krogh (Erik, L. L., et al., “A hidden Markov model for predictingtransmembrane helices in protein sequences,” Proc. of Sixth Int. Conf.on Intelligent Systems for Molecular Biology, pp. 175-182, Ed: J.Glasgow, et al., Menlo Park, Calif.: AAAI Press, 1998). This programindicates 85P1B3 to be an intracellular protein without transmembranedomains. These transmembrane prediction results are also summarized inTable XXV.

MODES OF CARRYING OUT THE INVENTION

I.) Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd.edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 85P1B3 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 85P1B3. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g.,a 85P1B3-related protein). For example an analog of the 85P1B3 proteincan be specifically bound by an antibody or T cell that specificallybinds to 85P1B3.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-85P1B3antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-85P1B3 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-85P1B3 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents the function of cells and/or causes destruction of cells. Theterm is intended to include radioactive isotopes chemotherapeuticagents, and toxins such as small molecule toxins or enzymatically activetoxins of bacterial, fungal, plant or animal origin, including fragmentsand/or variants thereof. Examples of cytotoxic agents include, but arenot limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin(PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin,gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,crotin, calicheamicin, sapaonaria officinalis inhibitor, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 85P1B3 gene orthat encode polypeptides other than 85P1B3 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 85P1B3 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 85P1B3 protein from cellularconstituents that are normally associated with the protein. A skilledartisan can readily employ standard purification methods to obtain anisolated 85P1B3 protein. Alternatively, an isolated protein can beprepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 85P1B3-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.,protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g., that is phosphorylated, glycosylated or amidated),or localization (e.g., secretory sequence, nuclear localizationsequence, etc.) or a sequence that is correlated with being immunogenic,either humorally or cellularly. A motif can be either contiguous orcapable of being aligned to certain positions that are generallycorrelated with a certain function or property. In the context of HLAmotifs, “motif” refers to the pattern of residues in a peptide ofdefined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs for HLA binding are typically different foreach protein encoded by each human HLA allele and differ in the patternof the primary and secondary anchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) (as shown for example in SEQ ID NO: 702) can alsobe uracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment, for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel,et al., Current Protocols in Molecular Biology, Wiley IntersciencePublishers (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook, et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 85P1B3 protein shown in FIG. 2 or FIG. 3).An analog is an example of a variant protein.

The 85P1B3-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 85P1B3 proteins orfragments thereof, as well as fusion proteins of a 85P1B3 protein and aheterologous polypeptide are also included. Such 85P1B3 proteins arecollectively referred to as the 85P1B3-related proteins, the proteins ofthe invention, or 85P1B3. The term “85P1B3-related protein” refers to apolypeptide fragment or an 85P1B3 protein sequence of 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or morethan 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70,80, 85, 90, 95, 100 or more than 100 amino acids.

II.) 85P1B3 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 85P1B3 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 85P1B3-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 85P1B3 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toan 85P1B3 gene, mRNA, or to an 85P1B3 encoding polynucleotide(collectively, “85P1B3 polynucleotides”). In all instances when referredto in this section, T can also be U in FIG. 2.

Embodiments of a 85P1B3 polynucleotide include: a 85P1B3 polynucleotidehaving the sequence shown in FIG. 2, the nucleotide sequence of 85P1B3as shown in FIG. 2, wherein T is U; at least 10 contiguous nucleotidesof a polynucleotide having the sequence as shown in FIG. 2; or, at least10 contiguous nucleotides of a polynucleotide having the sequence asshown in FIG. 2 where T is U. For example, embodiments of 85P1B3nucleotides comprise, without limitation:

(a) a polynucleotide comprising or consisting of the sequence as shownin FIG. 2 (SEQ ID NO: 727), wherein T can also be U;

(b) a polynucleotide comprising or consisting of the sequence as shownin FIG. 2 (SEQ ID NO: 727), from nucleotide residue number 13 throughnucleotide residue number 699, wherein T can also be U;

(c) a polynucleotide that encodes a 85P1B3-related protein whosesequence is encoded by the cDNAs contained in the plasmid designated______ deposited with American Type Culture Collection as Accession No.______;

(d) a polynucleotide that encodes an 85P1B3-related protein that is atleast 90% homologous to the entire amino acid sequence shown in (SEQ IDNO: 728);

(e) a polynucleotide that encodes an 85P1B3-related protein that is atleast 90% identical to the entire amino acid sequence shown in (SEQ IDNO: 728);

(f) a polynucleotide that encodes at least one peptide set forth inTables V-XVIII;

(g) a polynucleotide that encodes a peptide region of at least 5 aminoacids of FIG. 3 in any whole number increment up to 229 that includes anamino acid position having a value greater than 0.5 in theHydrophilicity profile of FIG. 5;

(h) a polynucleotide that encodes a peptide region of at least 5 aminoacids of FIG. 3 in any whole number increment up to 229 that includes anamino acid position having a value less than 0.5 in the Hydropathicityprofile of FIG. 6;

(i) a polynucleotide that encodes a peptide region of at least 5 aminoacids of FIG. 3 in any whole number increment up to 229 that includes anamino acid position having a value greater than 0.5 in the PercentAccessible Residues profile of FIG. 7;

(j) a polynucleotide that encodes a peptide region of at least 5 aminoacids of FIG. 3 in any whole number increment up to 229 that includes anamino acid position having a value greater than 0.5 in the AverageFlexibility profile on FIG. 8;

(k) a polynucleotide that encodes a peptide region of at least 5 aminoacids of FIG. 3 in any whole number increment up to 229 that includes anamino acid position having a value greater than 0.5 in the Beta-turnprofile of FIG. 9;

(l) a polynucleotide that is fully complementary to a polynucleotide ofany one of (a)-(k);

(m) a polynucleotide that selectively hybridizes under stringentconditions to a polynucleotide of (a)-(l); and

(n) a polynucleotide of any of (a)-(m) or peptide of (o) (seeimmediately below) together with a pharmaceutical excipient and/or in ahuman unit dose form.

Regarding item (n) immediately above, examples of embodiments of 85P1B3polypeptides comprise, without limitation:

(o) a peptide that is encoded by any of (a)-(k).

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 85P1B3polynucleotides that encode specific portions of the 85P1B3 mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 229 contiguousamino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 85P1B3 proteinshown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10to about amino acid 20 of the 85P1B3 protein shown in FIG. 2, or FIG. 3,polynucleotides encoding about amino acid 20 to about amino acid 30 ofthe 85P1B3 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 30 to about amino acid 40 of the 85P1B3 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 toabout amino acid 50 of the 85P1B3 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 50 to about amino acid 60 ofthe 85P1B3 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 60 to about amino acid 70 of the 85P1B3 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 toabout amino acid 80 of the 85P1B3 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 80 to about amino acid 90 ofthe 85P1B3 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 90 to about amino acid 100 of the 85P1B3 protein shownin FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending atthe carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3.Accordingly polynucleotides encoding portions of the amino acid sequence(of about 10 amino acids), of amino acids 100 through the carboxylterminal amino acid of the 85P1B3 protein are embodiments of theinvention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of the 85P1B3 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40, etc.) to aboutamino acid 20, (or 30, or 40 or 50, etc.) of the 85P1B3 protein shown inFIG. 2 or FIG. 3 can be generated by a variety of techniques well knownin the art. These polynucleotide fragments can include any portion ofthe 85P1B3 sequence as shown in FIG. 2 or FIG. 3.

Additional illustrative embodiments of the invention disclosed hereininclude 85P1B3 polynucleotide fragments encoding one or more of thebiological motifs contained within the 85P1B3 protein sequence,including one or more of the motif-bearing subsequences of the 85P1B3protein set forth in Tables V-XVIII. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 85P1B3 that exhibit homology to a known molecule. In anotherembodiment of the invention, typical polynucleotide fragments can encodeone or more of the 85P1B3 N-glycosylation sites, cAMP and cGMP-dependentprotein kinase phosphorylation sites, casein kinase II phosphorylationsites or N-myristoylation site and amidation sites.

II.A.) Uses of 85P1B3 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 85P1B3 gene maps to the chromosomallocation set forth in Example 3. For example, because the 85P1B3 genemaps to this chromosome, polynucleotides that encode different regionsof the 85P1B3 protein are used to characterize cytogenetic abnormalitiesof this chromosomal locale, such as abnormalities that are identified asbeing associated with various cancers. In certain genes, a variety ofchromosomal abnormalities including rearrangements have been identifiedas frequent cytogenetic abnormalities in a number of different cancers(see, e.g., Krajinovic, et al., Mutat. Res. (1998) 382:81-83; Johansson,et al., Blood (1995) 86:3905-3914 and Finger, et al., P.N.A.S. (1988)85:9158-9162). Thus, polynucleotides encoding specific regions of the85P1B3 protein provide new tools that can be used to delineate, withgreater precision than previously possible, cytogenetic abnormalities inthe chromosomal region that encodes 85P1B3 that may contribute to themalignant phenotype. In this context, these polynucleotides satisfy aneed in the art for expanding the sensitivity of chromosomal screeningin order to identify more subtle and less common chromosomalabnormalities (see, e.g., Evans, et al., Am. J. Obstet. Gynecol.(1994)171:1055-1057).

Furthermore, as 85P1B3 was shown to be highly expressed in prostate andother cancers, 85P1B3 polynucleotides are used in methods assessing thestatus of 85P1B3 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 85P1B3protein are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen, etc.) in specific regions of the 85P1B3 gene, suchas such regions containing one or more motifs. Exemplary assays includeboth RT-PCR assays as well as single-strand conformation polymorphism(SSCP) analysis (see, e.g., Marrogi, et al., J. Cutan. Pathol. (1999)26:369-378, both of which utilize polynucleotides encoding specificregions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 85P1B3. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNA's) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 85P1B3 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,85P1B3. See, for example, Jack Cohen, Oligodeoxynucleotides AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis (1988)1:1-5. The 85P1B3 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a non-bridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P., et al., J. Org. Chem. (1990) 55:4693-4698; andIyer, R. P., et al., J. Am. Chem. Soc. (1990) 112:1253-1254. Additional85P1B3 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge, et al., Antisense & Nucleic Acid Drug Development (1996)6:169-175).

The 85P1B3 antisense oligonucleotides of the present invention typicallycan be RNA or DNA that is complementary to and stably hybridizes withthe first 100 5′ codons or last 100 3′ codons of the 85P1B3 genomicsequence or the corresponding mRNA. Absolute complementarity is notrequired, although high degrees of complementarity are preferred. Use ofan oligonucleotide complementary to this region allows for the selectivehybridization to 85P1B3 mRNA and not to mRNA specifying other regulatorysubunits of protein kinase. In one embodiment, 85P1B3 antisenseoligonucleotides of the present invention are 15 to 30-mer fragments ofthe antisense DNA molecule that have a sequence that hybridizes to85P1B3 mRNA. Optionally, 85P1B3 antisense oligonucleotide is a 30-meroligonucleotide that is complementary to a region in the first 10 5′codons or last 10 3′ codons of 85P1B3. Alternatively, the antisensemolecules are modified to employ ribozymes in the inhibition of 85P1B3expression, see, e.g., Couture, L. A., et al., Trends Genet (1996)12:510-515.

II.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 85P1B3 polynucleotide in a sample and as ameans for detecting a cell expressing a 85P1B3 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 85P1B3 cDNA sequence shown in FIG. 2. Examples of primer pairscapable of specifically amplifying 85P1B3 mRNAs are also described inthe Examples. As will be understood by the skilled artisan, a great manydifferent primers and probes can be prepared based on the sequencesprovided herein and used effectively to amplify and/or detect a 85P1B3mRNA.

The 85P1B3 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 85P1B3 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 85P1B3 polypeptides; as tools for modulatingor inhibiting the expression of the 85P1B3 gene(s) and/or translation ofthe 85P1B3 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 85P1B3 or 85P1B3 related nucleic acid sequencefrom a naturally occurring source, such as humans or other mammals, aswell as the isolated nucleic acid sequence per se, which would compriseall or most of the sequences found in the probe used.

II.A.4.) Isolation of 85P1B3-Encoding Nucleic Acid Molecules

The 85P1B3 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding 85P1B3 gene product(s), as well as theisolation of polynucleotides encoding 85P1B3 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 85P1B3 gene product as well as polynucleotides that encode analogsof 85P1B3-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding an 85P1B3 gene arewell known (see, for example, Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology, Ausubel, et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 85P1B3gene cDNAs can be identified by probing with a labeled 85P1B3 cDNA or afragment thereof. For example, in one embodiment, the 85P1B3 cDNA (FIG.2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 85P1B3gene. The 85P1B3 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BAC's), yeastartificial chromosome libraries (YAC's), and the like, with 85P1B3 DNAprobes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 85P1B3 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids,YAC's, BAC's, as well as various viral and non-viral vectors well knownin the art, and cells transformed or transfected with such recombinantDNA or RNA molecules. Methods for generating such molecules are wellknown (see, for example, Sambrook, et al., 1989, supra). The inventionfurther provides a host-vector system comprising a recombinant DNAmolecule containing a 85P1B3 polynucleotide, fragment, analog orhomologue thereof within a suitable prokaryotic or eukaryotic host cell.Examples of suitable eukaryotic host cells include a yeast cell, a plantcell, or an animal cell, such as a mammalian cell or an insect cell(e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell).Examples of suitable mammalian cells include various prostate cancercell lines such as DU145 and TsuPrl, other transfectable or transducibleprostate cancer cell lines, primary cells (PrEC), as well as a number ofmammalian cells routinely used for the expression of recombinantproteins (e.g., COS, CHO, 293, 293T cells). More particularly, apolynucleotide comprising the coding sequence of 85P1B3 or a fragment,analog or homolog thereof can be used to generate 85P1B3 proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of85P1B3 proteins or fragments thereof are available, see for example,Sambrook, et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller, et al., MCB (1991) 11:1785). Using theseexpression vectors, 85P1B3 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1,NIH-3T3 and TsuPrl. The host-vector systems of the invention are usefulfor the production of a 85P1B3 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 85P1B3 and 85P1B3 mutations or analogs.

Recombinant human 85P1B3 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 85P1B3-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 85P1B3 or fragment,analog or homolog thereof, the 85P1B3 or related protein is expressed inthe 293T cells, and the recombinant 85P1B3 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-85P1B3 antibodies). In another embodiment, a 85P1B3 coding sequenceis subcloned into the retroviral vector pSRαMSVtkneo and used to infectvarious mammalian cell lines, such as NIH-3T3, TsuPrl, 293 and rat-1 inorder to establish 85P1B3 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the85P1B3 coding sequence can be used for the generation of a secreted formof recombinant 85P1B3 protein.

As discussed herein, redundancy in the genetic code permits variation in85P1B3 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol. (1989)9:5073-5080. Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak,PNAS (1995) 92:2662-2666, and Kozak, NAR (1987) 15:8125-8148).

III.) 85P1B3-Related Proteins

Another aspect of the present invention provides 85P1B3-relatedproteins. Specific embodiments of 85P1B3 proteins comprise a polypeptidehaving all or part of the amino acid sequence of human 85P1B3 as shownin FIG. 2 or FIG. 3. Alternatively, embodiments of 85P1B3 proteinscomprise variant, homolog or analog polypeptides that have alterationsin the amino acid sequence of 85P1B3 shown in FIG. 2 or FIG. 3.

In general, naturally occurring allelic variants of human 85P1B3 share ahigh degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of the 85P1B3 protein containconservative amino acid substitutions within the 85P1B3 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 85P1B3. One class of 85P1B3allelic variants are proteins that share a high degree of homology withat least a small region of a particular 85P1B3 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g., Table III herein; pages 13-15 Biochemistry,2^(nd) ED., Lubert Stryer, ed. (Stanford University); Henikoff, et al.,PNAS (1992) 89:10915-10919; Lei, et al., J. Biol. Chem.(1995)270:11882-11886).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 85P1B3 proteins such as polypeptideshaving amino acid insertions, deletions and substitutions. 85P1B3variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter, et al., Nucl. Acids Res. (1986)13:4331; Zoller, et al., Nucl. Acids Res. (1987) 10:6487), cassettemutagenesis (Wells, et al., Gene (1985) 34:315), restriction selectionmutagenesis (Wells, et al., Philos. Trans. R. Soc. London SerA (1986)317:415) or other known techniques can be performed on the cloned DNA toproduce the 85P1B3 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol. (1976) 150:1). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 85P1B3 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 85P1B3 protein having the amino acid sequence of SEQ IDNO: 703. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 85P1B3 variant alsospecifically binds to the 85P1B3 protein having the amino acid sequenceof SEQ ID NO: 703. A polypeptide ceases to be a variant of the proteinshown in SEQ ID NO: 703 when it no longer contains any epitope capableof being recognized by an antibody or T cell that specifically binds tothe 85P1B3 protein. Those skilled in the art understand that antibodiesthat recognize proteins bind to epitopes of varying size, and a groupingof the order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair, et al., J. Immunol. (2000) 165:6949-6955; Hebbes, et al.,Mol. Immunol. (1989) 26:865-873; Schwartz, et al., J. Immunol. (1985)135:2598-2608.

Another class of 85P1B3-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of SEQ ID NO:703 or a fragment thereof. Another specific class of 85P1B3 proteinvariants or analogs comprise one or more of the 85P1B3 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 85P1B3 fragments (nucleic or aminoacid) that have altered functional (e.g., immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of the85P1B3 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the85P1B3 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 85P1B3 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the 85P1B3protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of the 85P1B3 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of the 85P1B3 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofthe 85P1B3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of the 85P1B3 protein shownin FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of the 85P1B3 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofthe 85P1B3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of the 85P1B3 protein shownin FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of the 85P1B3 protein shown in FIG. 2 or FIG. 3,etc., throughout the entirety of the 85P1B3 amino acid sequence.Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or40, etc.) to about amino acid 20, (or 130, or 140 or 150, etc.) of the85P1B3 protein shown in FIG. 2 or FIG. 3 are embodiments of theinvention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

85P1B3-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 85P1B3-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofthe 85P1B3 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 85P1B3 polypeptides comprising the amino acid residues of one ormore of the biological motifs contained within the 85P1B3 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., Epimatrix™ andEpimer™, Brown University, and BIMAS).

Motif bearing subsequences of the 85P1B3 protein are set forth andidentified in Table XIX.

Table XX sets forth several frequently occurring motifs based on pfamsearches. The columns of Table XX list (1) motif name abbreviation, (2)percent identity found amongst the different member of the motif family,(3) motif name or description and (4) most common function; locationinformation is included if the motif is relevant for location.

Polypeptides comprising one or more of the 85P1B3 motifs discussed aboveare useful in elucidating the specific characteristics of a malignantphenotype in view of the observation that the 85P1B3 motifs discussedabove are associated with growth dysregulation and because 85P1B3 isoverexpressed in certain cancers (See, e.g., Table I). Casein kinase II,cAMP and camp-dependent protein kinase, and Protein Kinase C, forexample, are enzymes known to be associated with the development of themalignant phenotype (see, e.g., Chen, et al., Lab Invest. (1998)78:165-174; Gaiddon, et al., Endocrinology (1995) 136:4331-4338; Hall,et al., Nucleic Acids Research (1996) 24:1119-1126; Peterziel, et al.,Oncogene (1999) 18:6322-6329 and O'Brian, Oncol. Rep. (1998) 5:305-309).Moreover, both glycosylation and myristoylation are proteinmodifications also associated with cancer and cancer progression (see,e.g., Dennis, et al., Biochem. Biophys. Acta (1999) 1473:21-34; Raju, etal., Exp. Cell Res. (1997) 235:145-154). Amidation is another proteinmodification also associated with cancer and cancer progression (see,e.g., Treston, et al., J. Natl. Cancer Inst. Monogr (1992) 13:169-175).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 85P1B3 protein that are capable of optimally binding to specified HLAalleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, andBIMAS). Moreover, processes for identifying peptides that havesufficient binding affinity for HLA molecules and which are correlatedwith being immunogenic epitopes, are well known in the art, and arecarried out without undue experimentation. In addition, processes foridentifying peptides that are immunogenic epitopes, are well known inthe art, and are carried out without undue experimentation either invitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chesnut, et al.; Sette,Immunogenetics (1999) 50:201-212; Sette, et al., J. Immunol. (2001)166:1389-1397; Sidney, et al., Hum. Immunol. (1997) 58:12-20; Kondo, etal., Immunogenetics (1997) 45:249-258; Sidney, et al., J. Immunol.(1996) 157:3480-3490; and Falk, et al., Nature (1991) 351:290-296; Hunt,et al., Science (1992) 255:1261-1263; Parker, et al., J. Immunol. (1992)149:3580-3587; Parker, et al., J. Immunol. (1994) 152:163-175); Kast, W.M., et al., J. Immunol. (1994) 152: 3904-3912; Borras-Cuesta, et al.,Hum. Immunol. (2000) 61:266-278; Alexander, et al., J. Immunol. (2000)164:1625-1633; Alexander, et al., PMID: 7895164, UI: 95202582;O'Sullivan, et al., J. Immunol. (1991) 147:2663-2669; Alexander< et al.,Immunity (1994) 1:751-761 and Alexander, et al., Immunol. Res. (1998)18:79-92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

85P1B3-related proteins are embodied in many forms, preferably inisolated form. A purified 85P1B3 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 85P1B3 toantibody, T cell or other ligand. The nature and degree of isolation andpurification will depend on the intended use. Embodiments of a85P1B3-related proteins include purified 85P1B3-related proteins andfunctional, soluble 85P1B3-related proteins. In one embodiment, afunctional, soluble 85P1B3 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 85P1B3 proteins comprising biologicallyactive fragments of the 85P1B3 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the 85P1B3 protein, such asthe ability to elicit the generation of antibodies that specificallybind an epitope associated with the 85P1B3 protein; to be bound by suchantibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL.

85P1B3-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-85P1B3 antibodies, or T cells or in identifyingcellular factors that bind to 85P1B3.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 85P1B3 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site on theINTERNET; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, BrownUniversity, and BIMAS). Illustrating this, peptide epitopes from 85P1B3that are presented in the context of human MHC class I molecules HLA-A1,A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII).Specifically, the complete amino acid sequence of the 85P1B3 protein wasentered into the HLA Peptide Motif Search algorithm found in theBioinformatics and Molecular Analysis Section (BIMAS) web site listedabove. The HLA peptide motif search algorithm was developed by Dr. KenParker based on binding of specific peptide sequences in the groove ofHLA Class I molecules, in particular HLA-A2 (see, e.g., Falk, et al.,Nature (1991) 351:290-296; Hunt, et al., Science (1992) 255:1261-1263;Parker, et al., J. Immunol. (1992) 149:3580-3587; Parker, et al., J.Immunol. (1994) 152:163-175). This algorithm allows location and rankingof 8-mer, 9-mer, and 10-mer peptides from a complete protein sequencefor predicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker, et al., J. Immunol. (1992)149:3580-3587). Selected results of 85P1B3 predicted binding peptidesare shown in Tables V-XVIII herein. In Tables V-XVIII, the top 50ranking candidates, 9-mers and 10-mers, for each family member are shownalong with their location, the amino acid sequence of each specificpeptide, and an estimated binding score. The binding score correspondsto the estimated half time of dissociation of complexes containing thepeptide at 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue, et al., Prostate (1997) 30:73-78 and Peshwa, etal., Prostate (1998) 36:129-138). Immunogenicity of specific peptidescan be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV are to be “applied” to the 85P1B3 protein. As usedin this context “applied” means that the 85P1B3 protein is evaluated,e.g., visually or by computer-based patterns finding methods, asappreciated by those of skill in the relevant art. Every subsequence ofthe 85P1B3 of 8, 9, 10, or 11 amino acid residues that bears an HLAClass I motif, or a subsequence of 9 or more amino acid residues thatbear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 85P1B3-Related Proteins

In an embodiment described in the examples that follow, 85P1B3 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 85P1B3 with a C-terminal 6×His (SEQ ID NO: 709) and MYCtag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation,Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal thatcan be used to facilitate the production of a secreted 85P1B3 protein intransfected cells. The secreted HIS-tagged 85P1B3 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 85P1B3-Related Proteins

Modifications of 85P1B3-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 85P1B3polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 85P1B3. Another type of covalent modification of the 85P1B3polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 85P1B3 comprises linking the85P1B3 polypeptide to one of a variety of non-proteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 85P1B3-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 85P1B3 fused to another,heterologous polypeptide or amino acid sequence. Such a chimericmolecule can be synthesized chemically or recombinantly. A chimericmolecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 85P1B3 sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of85P1B3. A chimeric molecule can comprise a fusion of a 85P1B3-relatedprotein with a polyhistidine epitope tag, which provides an epitope towhich immobilized nickel can selectively bind, with cytokines or withgrowth factors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the 85P1B3. In an alternative embodiment, thechimeric molecule can comprise a fusion of a 85P1B3-related protein withan immunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 85P1B3polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 85P1B3-related Proteins

The proteins of the invention have a number of different specific uses.As 85P1B3 is highly expressed in prostate and other cancers,85P1B3-related proteins are used in methods that assess the status of85P1B3 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 85P1B3 protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations, etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting 85P1B3-relatedproteins comprising the amino acid residues of one or more of thebiological motifs contained within the 85P1B3 polypeptide sequence inorder to evaluate the characteristics of this region in normal versuscancerous tissues or to elicit an immune response to the epitope.Alternatively, 85P1B3-related proteins that contain the amino acidresidues of one or more of the biological motifs in the 85P1B3 proteinare used to screen for factors that interact with that region of 85P1B3.

85P1B3 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an85P1B3 protein), for identifying agents or cellular factors that bind to85P1B3 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 85P1B3 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 85P1B3 gene product.Antibodies raised against an 85P1B3 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 85P1B3protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 85P1B3-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 85P1B3 proteinsare used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 85P1B3-expressingcells (e.g., in radioscintigraphic imaging methods). 85P1B3 proteins arealso particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 85P1B3 Antibodies

Another aspect of the invention provides antibodies that bind to85P1B3-related proteins. Preferred antibodies specifically bind to a85P1B3-related protein and do not bind (or bind weakly) to peptides orproteins that are not 85P1B3-related proteins. For example, antibodiesbind 85P1B3 can bind 85P1B3-related proteins such as the homologs oranalogs thereof.

85P1B3 antibodies of the invention are particularly useful in prostatecancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 85P1B3 is alsoexpressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of85P1B3 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 85P1B3 and mutant 85P1B3-relatedproteins. Such assays can comprise one or more 85P1B3 antibodies capableof recognizing and binding a 85P1B3-related protein, as appropriate.These assays are performed within various immunological assay formatswell known in the art, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 85P1B3 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 85P1B3 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 85P1B3 expressingcancers such as prostate cancer.

85P1B3 antibodies are also used in methods for purifying a85P1B3-related protein and for isolating 85P1B3 homologues and relatedmolecules. For example, a method of purifying a 85P1B3-related proteincomprises incubating an 85P1B3 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a85P1B3-related protein under conditions that permit the 85P1B3 antibodyto bind to the 85P1B3-related protein; washing the solid matrix toeliminate impurities; and eluting the 85P1B3-related protein from thecoupled antibody. Other uses of the 85P1B3 antibodies of the inventioninclude generating anti-idiotypic antibodies that mimic the 85P1B3protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 85P1B3-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 85P1B3 canalso be used, such as a 85P1B3 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a85P1B3-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 85P1B3-related protein or 85P1B3 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly, et al., Ann. Rev. Immunol. (1997) 15: 617-648).

The amino acid sequence of 85P1B3 as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 85P1B3 protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses ofthe 85P1B3 amino acid sequence are used to identify hydrophilic regionsin the 85P1B3 structure. Regions of the 85P1B3 protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Thus, each region identified by any of theseprograms or methods is within the scope of the present invention.Methods for the generation of 85P1B3 antibodies are further illustratedby way of the examples provided herein. Methods for preparing a proteinor polypeptide for use as an immunogen are well known in the art. Alsowell known in the art are methods for preparing immunogenic conjugatesof a protein with a carrier, such as BSA, KLH or other carrier protein.In some circumstances, direct conjugation using, for example,carbodiimide reagents are used; in other instances linking reagents suchas those supplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 85P1B3 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

85P1B3 monoclonal antibodies can be produced by various means well knownin the art. For example, immortalized cell lines that secrete a desiredmonoclonal antibody are prepared using the standard hybridoma technologyof Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 85P1B3-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 85P1B3 protein can also be produced in the context of chimeric orcomplementarity determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 85P1B3 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDR's for corresponding human antibodysequences, are well known (see for example, Jones, et al. Nature (1986)321: 522-525; Riechmann, et al., Nature (1988) 332:323-327; Verhoeyen,et al., Science (1988) 239:1534-1536). See also, Carter, et al., Proc.Natl. Acad. Sci. USA (1993) 89:4285 and Sims, et al., J. Immunol. (1993)151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan, et al., NatureBiotechnology (1998) 16:535-539). Fully human 85P1B3 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, “Building an in vitro immune system: human antibodiesfrom phage display libraries,” Protein Engineering of Antibody Moleculesfor Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.),Nottingham Academic, pp. 45-64 (1993); Burton and Barbas, “HumanAntibodies from combinatorial libraries.” Id., pp. 65-82). Fully human85P1B3 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO 98/24893, Kucherlapati and Jakobovits, et al.,published Dec. 3, 1997 (see also, Jakobovits, Exp. Opin. Invest. Drugs(1998) 7:607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat.No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5Sep. 2000). This method avoids the in vitro manipulation required withphage display technology and efficiently produces high affinityauthentic human antibodies.

Reactivity of 85P1B3 antibodies with an 85P1B3-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,85P1B3-related proteins, 85P1B3-expressing cells or extracts thereof. A85P1B3 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more85P1B3 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff, et al., Cancer Res. (1993) 53:2560-2565).

V.) 85P1B3 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S., et al., Cell (1986)47:1071; Babbitt, B. P., et al., Nature (1985) 317:359; Townsend, A., etal., Annu. Rev. Immunol. (1989) 7:601; Germain, R. N., Annu. Rev.Immunol. (1993) 11:403). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. (1998) 160:3363; Rammensee, et al., Immunogenetics(1995) 41:178; Rammensee, et al., SYFPEITHI on the INTERNET; Sette, A.,et al., J Curr. Opin. Immunol. (1998) 10:478; Engelhard, V. H., Curr.Opin. Immunol. (1994) 6:13; Sette, A., et al., Curr. Opin. Immunol.(1992) 4:79; Sinigaglia, F., et al., J Curr. Biol. (1994) 6:52; Ruppert,et al., Cell (1993) 74:929-937; Kondo, et al., J. Immunol. (1995)155:4307-4312; Sidney, et al., J. Immunol. (1996) 157:3480-3490; Sidney,et al., Human Immunol. (1996) 45:79-93; Sette, A., et al., J.Immunogenetics (1999) 50:201-212, Review).

Furthermore, X-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R., Annu. Rev. Immunol. (1995) 13:587; Smith, et al., Immunity (1996)4:203; Fremont, et al., Immunity (1998) 8:305; Stern, et al., Structure(1994) 2:245; Jones, E. Y., Curr. Opin. Immunol. (1997) 9:75; Brown, J.H., et al., Nature (1993) 364:33; Guo, H. C., et al., Proc. Natl. Acad.Sci. USA (1993) 90:8053; Guo, H. C., et al., Nature (1992) 360:364;Silver, M. L., et al., Nature (1992) 360:367; Matsumura, M., et al.,Science (1992) 257:927; Madden, et al., Cell (1992) 70:1035; Fremont, D.H., et al., Science (1992) 257:919; Saper, M. A., et al., J. Mol. Biol.(1991) 219:277.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A., et al., Mol. Immunol. (1995) 32:603; Celis, E.,et al., Proc. Natl. Acad. Sci. USA (1994) 91:2105; Tsai, V., et al., J.Immunol. (1997) 158:1796; Kawashima, I., et al., Human Immunol. (1998)59:1). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A., etal., J. Immunol. (1996) 26:97; Wentworth, P. A., et al., Int. Immunol.(1996) 8:651; Alexander, J., et al., J. Immunol. (1997) 159:4753). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B., et al., J. Exp. Med. (1995)181:1047; Doolan, D. L., et al., Immunity (1997) 7:97; Bertoni, R., etal., J. Clin. Invest. (1997) 100:503; Threlkeld, S. C., et al., J.Immunol. (1997) 159:1648; Diepolder, H. M., et al., J. Virol. (1997)71:6011). Accordingly, recall responses are detected by culturing PBLfrom subjects that have been exposed to the antigen due to disease andthus have generated an immune response “naturally”, or from patients whowere vaccinated against the antigen. PBL from subjects are cultured invitro for 1-2 weeks in the presence of test peptide plus antigenpresenting cells (APC) to allow activation of “memory” T cells, ascompared to “naive” T cells. At the end of the culture period, T cellactivity is detected using assays including ⁵¹Cr release involvingpeptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 85P1B3 Transgenic Animals

Nucleic acids that encode a 85P1B3-related protein can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 85P1B3 can be used to clone genomic DNA that encodes 85P1B3.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 85P1B3. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 85P1B3 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 85P1B3can be used to examine the effect of increased expression of DNA thatencodes 85P1B3. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 85P1B3 can be used to construct a85P1B3 “knock out” animal that has a defective or altered gene encoding85P1B3 as a result of homologous recombination between the endogenousgene encoding 85P1B3 and altered genomic DNA encoding 85P1B3 introducedinto an embryonic cell of the animal. For example, cDNA that encodes85P1B3 can be used to clone genomic DNA encoding 85P1B3 in accordancewith established techniques. A portion of the genomic DNA encoding85P1B3 can be deleted or replaced with another gene, such as a geneencoding a selectable marker that can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector (see, e.g., Thomas, et al., Cell(1987) 51:503 for a description of homologous recombination vectors).The vector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see, e.g., Li, et al.,Cell (1992) 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal, and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 85P1B3 polypeptide.

VII.) Methods for the Detection of 85P1B3

Another aspect of the present invention relates to methods for detecting85P1B3 polynucleotides and 85P1B3-related proteins, as well as methodsfor identifying a cell that expresses 85P1B3. The expression profile of85P1B3 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 85P1B3 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 85P1B3 geneproducts in patient samples can be analyzed by a variety protocols thatare well known in the art including immunohistochemical analysis, thevariety of Northern blotting techniques including in situ hybridization,RT-PCR analysis (for example on laser capture micro-dissected samples),Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of85P1B3 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 85P1B3 polynucleotides include, for example, a 85P1B3gene or fragment thereof, 85P1B3 mRNA, alternative splice variant 85P1B3mRNAs, and recombinant DNA or RNA molecules that contain a 85P1B3polynucleotide. A number of methods for amplifying and/or detecting thepresence of 85P1B3 polynucleotides are well known in the art and can beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 85P1B3 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using an85P1B3 polynucleotides as sense and antisense primers to amplify 85P1B3cDNAs therein; and detecting the presence of the amplified 85P1B3 cDNA.Optionally, the sequence of the amplified 85P1B3 cDNA can be determined.

In another embodiment, a method of detecting a 85P1B3 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 85P1B3 polynucleotides assense and antisense primers; and detecting the presence of the amplified85P1B3 gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequence provided forthe 85P1B3 (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an85P1B3 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 85P1B3-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 85P1B3-related proteinin a biological sample comprises first contacting the sample with a85P1B3 antibody, a 85P1B3-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 85P1B3 antibody; andthen detecting the binding of 85P1B3-related protein in the sample.

Methods for identifying a cell that expresses 85P1B3 are also within thescope of the invention. In one embodiment, an assay for identifying acell that expresses a 85P1B3 gene comprises detecting the presence of85P1B3 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 85P1B3 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 85P1B3, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 85P1B3 gene comprises detecting the presence of 85P1B3-related proteinin the cell or secreted by the cell. Various methods for the detectionof proteins are well known in the art and are employed for the detectionof 85P1B3-related proteins and cells that express 85P1B3-relatedproteins.

85P1B3 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 85P1B3 gene expression. For example,85P1B3 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 85P1B3 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies 85P1B3expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 85P1B3-Related Genes andtheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers, et al., Lab Invest. (1997) 77:437-438 and Isaacs, et al.,Cancer Surv. (1995) 23:19-32). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant 85P1B3expression in cancers) allows for early detection of such aberrantphysiology, before a pathologic state such as cancer has progressed to astage that therapeutic options are more limited and or the prognosis isworse. In such examinations, the status of 85P1B3 in a biological sampleof interest can be compared, for example, to the status of 85P1B3 in acorresponding normal sample (e.g., a sample from that individual oralternatively another individual that is not affected by a pathology).An alteration in the status of 85P1B3 in the biological sample (ascompared to the normal sample) provides evidence of dysregulatedcellular growth. In addition to using a biological sample that is notaffected by a pathology as a normal sample, one can also use apredetermined normative value such as a predetermined normal level ofmRNA expression (see, e.g., Grever, et al., J. Comp. Neurol. (1996)376:306-314 and U.S. Pat. No. 5,837,501) to compare 85P1B3 status in asample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 85P1B3 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 85P1B3 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 85P1B3 comprises a change in the location of 85P1B3 and/or85P1B3 expressing cells and/or an increase in 85P1B3 mRNA and/or proteinexpression.

85P1B3 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 85P1B3 geneand gene products are found, for example in Ausubel, et al., eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 85P1B3 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in the85P1B3 gene), Northern analysis and/or PCR analysis of 85P1B3 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 85P1B3 mRNAs), and Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 85P1B3 proteins and/orassociations of 85P1B3 proteins with polypeptide binding partners).Detectable 85P1B3 polynucleotides include, for example, a 85P1B3 gene orfragment thereof, 85P1B3 mRNA, alternative splice variants, 85P1B3mRNAs, and recombinant DNA or RNA molecules containing a 85P1B3polynucleotide.

The expression profile of 85P1B3 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of85P1B3 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 85P1B3 status anddiagnosing cancers that express 85P1B3, such as cancers of the tissueslisted in Table I. For example, because 85P1B3 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 85P1B3 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 85P1B3 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 85P1B3 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 85P1B3 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 85P1B3 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 85P1B3 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 85P1B3 expressing cells (e.g., those that express85P1B3 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 85P1B3-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of85P1B3 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy, et al., Prostate (2000) 42:315-317; Su, et al.,Semin. Surg. Oncol. (2000) 18:17-28 and Freeman, et al., J. Urol. (1995)154:474-478).

In one aspect, the invention provides methods for monitoring 85P1B3 geneproducts by determining the status of 85P1B3 gene products expressed bycells from an individual suspected of having a disease associated withdysregulated cell growth (such as hyperplasia or cancer) and thencomparing the status so determined to the status of 85P1B3 gene productsin a corresponding normal sample. The presence of aberrant 85P1B3 geneproducts in the test sample relative to the normal sample provides anindication of the presence of dysregulated cell growth within the cellsof the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 85P1B3 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 85P1B3 mRNA can, for example, beevaluated in tissue samples including but not limited to those listed inTable I. The presence of significant 85P1B3 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 85P1B3mRNA or express it at lower levels.

In a related embodiment, 85P1B3 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 85P1B3 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 85P1B3 expressed in a corresponding normal sample. In one embodiment,the presence of 85P1B3 protein is evaluated, for example, usingimmunohistochemical methods. 85P1B3 antibodies or binding partnerscapable of detecting 85P1B3 protein expression are used in a variety ofassay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 85P1B3nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi, et al., J. Cutan. Pathol. (1999) 26:369-378). For example, amutation in the sequence of 85P1B3 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 85P1B3 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 85P1B3 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 85P1B3 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo, et al., Am. J.Pathol. (1999) 155:1985-1992). In addition, this alteration is presentin at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks, et al., Cancer Epidemiol. Biomarkers Prev.(1998) 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe, et al., Int. J. Cancer (1998) 76:903-908). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymeswhich cannot cleave sequences that contain methylated CpG sites toassess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubel, et al., eds., 1995.

Gene amplification is an additional method for assessing the status of85P1B3. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA(1980) 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 85P1B3 expression. The presence of RT-PCR amplifiable85P1B3 mRNA provides an indication of the presence of cancer. RT-PCRassays are well known in the art. RT-PCR detection assays for tumorcells in peripheral blood are currently being evaluated for use in thediagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik, et al., Urol. Res. (1997)25:373-384; Ghossein, et al., J. Clin. Oncol. (1995)13:1195-2000;Heston, et al., Clin. Chem. (1995)41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting85P1B3 mRNA or 85P1B3 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 85P1B3 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 85P1B3 in prostate or other tissue isexamined, with the presence of 85P1B3 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 85P1B3 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 85P1B3 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 85P1B3 mRNA or 85P1B3 proteinexpressed by tumor cells, comparing the level so determined to the levelof 85P1B3 mRNA or 85P1B3 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 85P1B3 mRNA or 85P1B3 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 85P1B3 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 85P1B3 nucleotide and amino acid sequences in a biological sample, inorder to identify perturbations in the structure of these molecules suchas insertions, deletions, substitutions and the like. The presence ofone or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 85P1B3 mRNA or85P1B3 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 85P1B3 mRNA or 85P1B3 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 85P1B3 mRNA or 85P1B3 proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining 85P1B3 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 85P1B3nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 85P1B3 gene and 85P1B3gene products (or perturbations in 85P1B3 gene and 85P1B3 gene products)and a factor that is associated with malignancy, as a means fordiagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g., PSA, PSCA andPSM expression for prostate cancer, etc.) as well as gross cytologicalobservations (see, e.g., Bocking, et al., Anal. Quant. Cytol. (1984)6:74-88; Epstein, Hum. Pathol. (1995) 26:223-229; Thorson, et al., Mod.Pathol. (1998) 11:543-551; Baisden, et al., Am. J. Surg. Pathol. (1999)23:918-924). Methods for observing a coincidence between the expressionof 85P1B3 gene and 85P1B3 gene products (or perturbations in 85P1B3 geneand 85P1B3 gene products) and another factor that is associated withmalignancy are useful, for example, because the presence of a set ofspecific factors that coincide with disease provides information crucialfor diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 85P1B3 gene and 85P1B3 gene products (or perturbations in85P1B3 gene and 85P1B3 gene products) and another factor associated withmalignancy entails detecting the overexpression of 85P1B3 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 85P1B3 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 85P1B3 and PSA mRNA in prostate tissue is examined,where the coincidence of 85P1B3 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 85P1B3 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification-technologies are well known in the art.Standard methods for the detection and quantification of 85P1B3 mRNAinclude in situ hybridization using labeled 85P1B3 riboprobes, Northernblot and related techniques using 85P1B3 polynucleotide probes, RT-PCRanalysis using primers specific for 85P1B3, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR is used todetect and quantify 85P1B3 mRNA expression. Any number of primerscapable of amplifying 85P1B3 can be used for this purpose, including butnot limited to the various primer sets specifically described herein. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type 85P1B3 protein can be used in animmunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 85P1B3

The 85P1B3 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 85P1B3, as well as pathways activated by 85P1B3 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature (1999) 402:83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 85P1B3 protein sequences. In such methods, peptidesthat bind to 85P1B3 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the85P1B3 protein.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 85P1B3 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 85P1B3 are used to identifyprotein-protein interactions mediated by 85P1B3. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton,B. J., et al., Biochem. Biophys. Res. Commun. (1999) 261:646-651).85P1B3 protein can be immunoprecipitated from 85P1B3-expressing celllines using anti-85P1B3 antibodies. Alternatively, antibodies againstHis-tag can be used in a cell line engineered to express fusions of85P1B3 and a His-tag (vectors mentioned above). The immunoprecipitatedcomplex can be examined for protein association by procedures such asWestern blotting, ³⁵S-methionine labeling of proteins, proteinmicrosequencing, silver staining and two-dimensional gelelectrophoresis.

Small molecules and ligands that interact with 85P1B3 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 85P1B3's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 85P1B3-related ion channel, protein pump, or cell communicationfunctions are identified and used to treat patients that have a cancerthat expresses 85P1B3 (see, e.g., Hille, B., Ionic Channels of ExcitableMembranes, 2^(nd) Ed., Sinauer Assoc., Sunderland, Mass., 1992).Moreover, ligands that regulate 85P1B3 function can be identified basedon their ability to bind 85P1B3 and activate a reporter construct.Typical methods are discussed for example in U.S. Pat. No. 5,928,868issued 27 Jul. 1999, and include methods for forming hybrid ligands inwhich at least one ligand is a small molecule. In an illustrativeembodiment, cells engineered to express a fusion protein of 85P1B3 and aDNA-binding protein are used to co-express a fusion protein of a hybridligand/small molecule and a cDNA library transcriptional activatorprotein. The cells further contain a reporter gene, the expression ofwhich is conditioned on the proximity of the first and second fusionproteins to each other, an event that occurs only if the hybrid ligandbinds to target sites on both hybrid proteins. Those cells that expressthe reporter gene are selected and the unknown small molecule or theunknown ligand is identified. This method provides a means ofidentifying modulators which activate or inhibit 85P1B3.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 85P1B3 amino acid sequence shown in FIG.2 or FIG. 3, comprising the steps of contacting a population ofmolecules with the 85P1B3 amino acid sequence, allowing the populationof molecules and the 85P1B3 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 85P1B3 amino acid sequence, and thenseparating molecules that do not interact with the 85P1B3 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 85P1B3 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 85P1B3. In apreferred embodiment, the 85P1B3 amino acid sequence is contacted with alibrary of peptides.

X.) Therapeutic Methods and Compositions

The identification of 85P1B3 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 85P1B3 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the85P1B3 protein are useful for patients suffering from a cancer thatexpresses 85P1B3. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the 85P1B3 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of the 85P1B3 gene or translation of 85P1B3mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 85P1B3-relatedprotein or 85P1B3-related nucleic acid. In view of the expression of85P1B3, cancer vaccines prevent and/or treat 85P1B3-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge, et al., Int. J. Cancer (1995) 63:231-237; Fong, et al., J.Immunol. (1997) 159:3113-3117).

Such methods can be readily practiced by employing a 85P1B3-relatedprotein, or an 85P1B3-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 85P1B3 immunogen (whichtypically comprises a number of antibody or T cell epitopes). Skilledartisans understand that a wide variety of vaccine systems for deliveryof immunoreactive epitopes are known in the art (see, e.g., Heryln, etal., Ann Med (1999) 31:66-78; Maruyama, et al., Cancer ImmunolImmunother (2000) 49:123-132) Briefly, such methods of generating animmune response (e.g., humoral and/or cell-mediated) in a mammal,comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g., an epitope present in the 85P1B3 proteinshown in SEQ ID NO: 703 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.,generates antibodies that specifically recognize that epitope). In apreferred method, the 85P1B3 immunogen contains a biological motif, seee.g., Tables V-XVIII, or a peptide of a size range from 85P1B3 indicatedin FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 85P1B3 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A., et al., J.Clin. Invest. (1995) 95:341), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. (1991) 28:287-294; Alonso, et al., Vaccine(1994) 12:299-306; Jones, et al., Vaccine (1995) 13:675-681), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi, et al., Nature (1990) 344:873-875; Hu, et al., Clin ExpImmunol. (1998) 113:235-243), multiple antigen peptide systems (MAP's)(see, e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. (1988)85:5409-5413; Tam, J. P., J. Immunol. Methods (1996) 196:17-32),peptides formulated as multivalent peptides; peptides for use inballistic delivery systems, typically crystallized peptides, viraldelivery vectors (Perkus, M. E., et al., Concepts in vaccinedevelopment, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S., etal., Nature (1986) 320:535; Hu, S. L., et al., Nature (1986) 320:537;Kieny, M.-P., et al., AIDS Bio/Technology (1986) 4:790; Top, F. H., etal., J. Infect. Dis. (1971) 124:148; Chanda, P. K., et al., Virology(1990) 175:535), particles of viral or synthetic origin (e.g., Kofler,N., et al., J. Immunol. Methods. (1996) 192:25; Eldridge, J. H., et al.,Sem. Hematol. (1993) 30:16; Falo, Jr., L. D., et al., Nature Med. (1995)7:649), adjuvants (Warren, H. S., et al., Annu. Rev. Immunol. (1986)4:369; Gupta, R. K., et al., Vaccine (1993) 11:293), liposomes (Reddy,R., et al., J. Immunol. (1992) 148:1585; Rock, K. L., Immunol. Today(1996) 17:131), or, naked or particle absorbed cDNA (Ulmer, J. B., etal., Science (1993) 259:1745; Robinson, H. L., et al., Vaccine (1993)11:957; Shiver, J. W., et al., Concepts in vaccine development,Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., et al., Annu. Rev.Immunol. (1994) 12:923, and Eldridge, J. H., et al., Sem. Hematol.(1993) 30:16). Toxin-targeted delivery technologies, also known asreceptor mediated targeting, such as those of Avant Immunotherapeutics,Inc. (Needham, Mass.) may also be used.

In patients with 85P1B3-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 85P1B3 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University, and, BIMAS).In a preferred embodiment, the 85P1B3 immunogen contains one or moreamino acid sequences identified using techniques well known in the art,such as the sequences shown in Tables V-XVIII or a peptide of 8, 9, 10or 11 amino acids specified by an HLA Class I motif/supermotif (e.g.,Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of atleast 9 amino acids that comprises an HLA Class II motif/supermotif(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, theHLA Class I binding groove is essentially closed ended so that peptidesof only a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a peptide of about 9 or more amino acids can be bound by anHLA Class II molecule. Due to the binding groove differences between HLAClass I and II, HLA Class I motifs are length specific, i.e., positiontwo of a Class I motif is the second amino acid in an amino to carboxyldirection of the peptide. The amino acid positions in a Class II motifare relative only to each other, not the overall peptide, i.e.,additional amino acids can be attached to the amino and/or carboxyltermini of a motif-bearing sequence. HLA Class II epitopes are often 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g., the 85P1B3 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 85P1B3 in a host, by contacting the host with asufficient amount of at least one 85P1B3B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 85P1B3B cell or cytotoxic T-cell epitopeor analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 85P1B3-related protein or aman-made multiepitopic peptide comprising: administering 85P1B3immunogen (e.g., the 85P1B3 protein or a peptide fragment thereof, an85P1B3 fusion protein or analog, etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander, et al., J. Immunol. (2000)164:1625-1633; Alexander, et al., Immunity (1994) 1:751-761 andAlexander, et al., Immunol. Res. (1998) 18:79-92). An alternative methodcomprises generating an immune response in an individual against a85P1B3 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 85P1B3 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 85P1B3. Constructscomprising DNA encoding a 85P1B3-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 85P1B3 protein/immunogen.Alternatively, a vaccine comprises a 85P1B3-related protein. Expressionof the 85P1B3-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear 85P1B3 protein. Various prophylactic and therapeutic geneticimmunization techniques known in the art can be used (for review, seeinformation and references published on the INTERNET). Nucleicacid-based delivery is described, for instance, in Wolff, et al.,Science (1990) 247:1465 as well as U.S. Pat. Nos. 5,580,859; 5,589,466;5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples ofDNA-based delivery technologies include “naked DNA”, facilitated(bupivicaine, polymers, peptide-mediated) delivery, cationic lipidcomplexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and Sindbis virus (see, e.g., Restifo, Curr. Opin. Immunol. (1996)8:658-663; Tsang, et al., J. Natl. Cancer Inst. (1995) 87:982-990).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 85P1B3-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover, et al.,Nature (1991) 351:456-460. A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 85P1B3-related nucleicacid molecule. In one embodiment, the full-length human 85P1B3 cDNA isemployed. In another embodiment, 85P1B3 nucleic acid molecules encodingspecific cytotoxic T lymphocyte (CTL) and/or antibody epitopes areemployed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APC's) such as dendritic cells (DC) to present 85P1B3 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa, et al., Prostate (1996) 28:65-69;Murphy, et al., Prostate (1996) 29:371-380). Thus, dendritic cells canbe used to present 85P1B3 peptides to T cells in the context of MHCclass I or II molecules. In one embodiment, autologous dendritic cellsare pulsed with 85P1B3 peptides capable of binding to MHC class I and/orclass II molecules. In another embodiment, dendritic cells are pulsedwith the complete 85P1B3 protein. Yet another embodiment involvesengineering the overexpression of the 85P1B3 gene in dendritic cellsusing various implementing vectors known in the art, such as adenovirus(Arthur, et al., Cancer Gene Ther. (1997) 4:17-25), retrovirus(Henderson, et al., Cancer Res. (1996) 56:3763-3770), lentivirus,adeno-associated virus, DNA transfection (Ribas, et al., Cancer Res.(1997) 57:2865-2869), or tumor-derived RNA transfection (Ashley, et al.,J. Exp. Med (1997) 186:1177-1182). Cells that express 85P1B3 can also beengineered to express immune modulators, such as GM-CSF, and used asimmunizing agents.

X.B.) 85P1B3 as a Target for Antibody-Based Therapy

85P1B3 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 85P1B3 is expressed by cancer cells of various lineages relativeto corresponding normal cells, systemic administration of85P1B3-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 85P1B3 areuseful to treat 85P1B3-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

85P1B3 antibodies can be introduced into a patient such that theantibody binds to 85P1B3 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 85P1B3,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 85P1B3 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers, et al., Blood (1999)93:3678-3684). When cytotoxic and/or therapeutic agents are delivereddirectly to cells, such as by conjugating them to antibodies specificfor a molecule expressed by that cell (e.g., 85P1B3), the cytotoxicagent will exert its known biological effect (i.e., cytotoxicity) onthose cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g., an anti-85P1B3 antibody) that binds to a marker (e.g., 85P1B3)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 85P1B3, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a85P1B3 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-85P1B3 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen, et al., Crit. Rev. Immunol. (1998) 18:133-138),multiple myeloma (Ozaki, et al., Blood (1997) 90:3179-3186, Tsunenari,et al., Blood (1997) 90:2437-2444), gastric cancer (Kasprzyk, et al.,Cancer Res. (1992) 52:2771-2776), B-cell lymphoma (Funakoshi, et al., JImmunother. Emphasis Tumor Immunol. (1996) 19:93-101), leukemia (Zhong,et al., Leuk. Res. (1996) 20:581-589), colorectal cancer (Moun, et al.,Cancer Res. (1994) 54:6160-6166; Velders, et al., Cancer Res. (1995)55:4398-4403), and breast cancer (Shepard, et al., J. Clin. Immunol.(1991) 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of Y⁹¹ or I¹³¹ toanti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. orBexxar™, Coulter Pharmaceuticals), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treatprostate cancer, for example, 85P1B3 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation.

Although 85P1B3 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 85P1B3expression, preferably using immunohistochemical assessments of tumortissue, quantitative 85P1B3 imaging, or other techniques that reliablyindicate the presence and degree of 85P1B3 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-85P1B3 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-85P1B3monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-85P1B3 mAbs that exert adirect biological effect on tumor growth are useful to treat cancersthat express 85P1B3. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-85P1B3 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 85P1B3antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-85P1B3 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-85P1B3 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-85P1B3 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-85P1B3 antibody formulations are administered via any route capableof delivering the antibodies to a tumor cell. Routes of administrationinclude, but are not limited to, intravenous, intraperitoneal,intramuscular, intratumor, intradermal, and the like. Treatmentgenerally involves repeated administration of the anti-85P1B3 antibodypreparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1 to about 10 mg/kg body weight. In general, doses in the range of10-500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-85P1B3 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 85P1B3 expression in the patient, the extent ofcirculating shed 85P1B3 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 85P1B3 in agiven sample (e.g. the levels of circulating 85P1B3 antigen and/or85P1B3 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt™ levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-85P1B3 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 85P1B3-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-85P1B3 antibodiesthat mimic an epitope on a 85P1B3-related protein (see, for example,Wagner, et al., Hybridoma (1997) 16:33-40; Foon, et al., Clin. Invest.(1995) 96:334-342; Herlyn, et al., Cancer Immunol. Immunother. (1996)43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 85P1B3 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTL's thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-5-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (See, e.g.,Davila, et al., J. Immunol. (2000) 165:539-547.)

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTL'sand/or HTL's specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 85P1B3 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described, e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg, et al., Science (1997) 278:1447-1450). Epitopes fromone TAA may be used in combination with epitopes from one or moreadditional TAA's to produce a vaccine that targets tumors with varyingexpression patterns of frequently-expressed TAA's.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minipene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka,et al., J. Immunol. (1999) 162:3915-3925; An, L., et al., J. Virol.(1997) 71:2292; Thomson, S. A., et al., J. Immunol. (1996) 157:822;Whitton, J. L., et al., J. Virol. (1993) 67:348; Hanke, R., et al.,Vaccine (1998) 16:426. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 85P1B3, the PADRE®universal helper T cell epitope (or multiple HTL epitopes from 85P1B3),and an endoplasmic reticulum-translocating signal sequence can beengineered. A vaccine may also comprise epitopes that are derived fromother TAA's.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTL'srecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g., poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g., ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISS's or CpG's) appear to playa role in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bicistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g., TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino, et al., BioTechniques (1988) 6:682;U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'lAcad. Sci. USA (1987) 84:7413. In addition, peptides and compoundsreferred to collectively as protective, interactive, non-condensingcompounds (PINC) could also be complexed to purified plasmid DNA toinfluence variables such as stability, intramuscular dispersion, ortrafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and re-stimulated for one weekin the presence of peptides encoding each epitope being tested.Thereafter, for CTL effector cells, assays are conducted for cytolysisof peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTL's. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:710), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 711), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 712). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 713), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε-and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-5-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature (1989)342:561). Peptides of the invention can be coupled to P₃CSS, forexample, and the lipopeptide administered to an individual tospecifically prime an immune response to the target antigen. Moreover,because the induction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DC's which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 85P1B3. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 85P1B3.

X.D. Adoptive Immunotherapy

Antigenic 85P1B3-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses85P1B3. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 85P1B3. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 85P1B3-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTL's or pulsed dendriticcells) delivered to the patient may vary according to the stage of thedisease or the patient's health status. For example, in a patient with atumor that expresses 85P1B3, a vaccine comprising 85P1B3-specific CTLmay be more efficacious in killing tumor cells in patient with advanceddisease than alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g., as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu. For antibodies, a treatment generally involves repeatedadministration of the anti-85P1B3 antibody preparation, via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. In general, doses in the range of 10-500 mg mAb per week areeffective and well tolerated. Moreover, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-85P1B3 mAb preparation represents anacceptable dosing regimen. As appreciated by those of skill in the art,various factors can influence the ideal dose in a particular case. Suchfactors include, for example, half life of a composition, the bindingaffinity of an Ab, the immunogenicity of a substance, the degree of85P1B3 expression in the patient, the extent of circulating shed 85P1B3antigen, the desired steady-state concentration level, frequency oftreatment, and the influence of chemotherapeutic or other agents used incombination with the treatment method of the invention, as well as thehealth status of a particular patient.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 80, 100, 200, 300, 400 or 500 mg/kg up to an independentlyselected upper limit, greater than the lower limit, of about 60, 80,100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about anyof the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg,0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally,parenteral routes of administration may require higher doses ofpolynucleotide compared to more direct application to the nucleotide todiseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. (1980) 9:467, and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc., in a dose which variesaccording to, inter alia, the manner of administration, the peptidebeing delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 85P1B3.

As disclosed herein, 85P1B3 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

85P1B3 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill, et al., J Urol. (2000) 163:503-5120; Polascik, et al., J Urol.(1999) 162:293-306 and Fortier, et al., J Nat. Cancer Inst. (1999)91:1635-1640). A variety of other diagnostic markers are also used insimilar contexts including p53 and K-ras (see, e.g., Tulchinsky, et al.,Int J Mol Med (1999) 4:99-102 and Minimoto, et al., Cancer Detect Prev(2000) 24:1-12). Therefore, this disclosure of the 85P1B3polynucleotides and polypeptides (as well as the 85P1B3 polynucleotideprobes and anti-85P1B3 antibodies used to identify the presence of thesemolecules) and their properties allows skilled artisans to utilize thesemolecules in methods that are analogous to those used, for example, in avariety of diagnostic assays directed to examining conditions associatedwith cancer.

Typical embodiments of diagnostic methods which utilize the 85P1B3polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief, et al., Biochem.Mol. Biol. Int. (1994)33:567-574) and primers (for example in PCRanalysis, see, e.g., Okegawa, et al., J. Urol. (2000) 163:1189-1190) toobserve the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the85P1B3 polynucleotides described herein can be utilized in the same wayto detect 85P1B3 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan, et al., Urology(2000) 55:560-563) or the metastasis of prostate cells (see, e.g.,Alanen, et al., Pathol. Res. Pract. (1996) 192:233-237), the 85P1B3polypeptides described herein can be utilized to generate antibodies foruse in detecting 85P1B3 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland, etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 85P1B3polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 85P1B3-expressing cells (lymph node) is found tocontain 85P1B3-expressing cells such as the 85P1B3 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 85P1B3 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 85P1B3 or express 85P1B3 at adifferent level are found to express 85P1B3 or have an increasedexpression of 85P1B3 (see, e.g., the 85P1B3 expression in the cancerslisted in Table I and in patient samples, etc., shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 85P1B3) such as PSA, PSCA, etc., (see, e.g., Alanen, et al.,Pathol. Res. Pract. (1996) 192:233-237).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,85P1B3 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.,Biotechniques (1998) 25:472-476, 478-480; Robertson, et al., MethodsMol. Biol. (1998) 98:121-154). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 85P1B3 polynucleotidefragment is used as a probe to show the expression of 85P1B3 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai, et al., Fetal Diagn. Ther.(1996) 11:407-413 and Current Protocols In Molecular Biology, Volume 2,Unit 2, Frederick M. Ausubel, et al., eds. (1995)). Polynucleotidefragments and variants are useful in this context where they are capableof binding to a target polynucleotide sequence (e.g., the 85P1B3polynucleotide shown in SEQ ID NO: 701) under conditions of highstringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 85P1B3 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel,et al., eds. (1995)). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 85P1B3biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g., the 85P1B3 polypeptide shown in SEQ ID NO:703).

As shown herein, the 85P1B3 polynucleotides and polypeptides (as well asthe 85P1B3 polynucleotide probes and anti-85P1B3 antibodies or T cellsused to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of 85P1B3gene products, in order to evaluate the presence or onset of a diseasecondition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen, et al., Pathol. Res. Pract.(1996) 192:233-237), and consequently, materials such as 85P1B3polynucleotides and polypeptides (as well as the 85P1B3 polynucleotideprobes and anti-85P1B3 antibodies used to identify the presence of thesemolecules) must be employed to confirm metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 85P1B3polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 85P1B3 gene maps(see Example 3 below). Moreover, in addition to their use in diagnosticassays, the 85P1B3-related proteins and polynucleotides disclosed hereinhave other utilities such as their use in the forensic analysis oftissues of unknown origin (see, e.g., Takahama, K., Forensic Sci Int(1996) 80:63-69).

Additionally, 85P1B3-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 85P1B3. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to the 85P1B3 antigen. Antibodies orother molecules that react with 85P1B3 can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 85P1B3 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 85P1B3 to its binding partner or its association withother protein(s) as well as methods for inhibiting 85P1B3 function.

XII.A.) Inhibition of 85P1B3 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 85P1B3 are introduced into 85P1B3expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-85P1B3 antibody is expressed intracellularly,binds to 85P1B3 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson, et al., Proc.Natl. Acad. Sci. USA (1995) 92:3137-3141; Beerli, et al., J. Biol. Chem.(1994) 289:23931-23936; Deshane, et al., Gene Ther. (1994) 1:332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL (SEQ ID NO: 708) amino acid motif. Intrabodies intended to exertactivity in the nucleus are engineered to include a nuclear localizationsignal. Lipid moieties are joined to intrabodies in order to tether theintrabody to the cytosolic side of the plasma membrane. Intrabodies canalso be targeted to exert function in the cytosol. For example,cytosolic intrabodies are used to sequester factors within the cytosol,thereby preventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 85P1B3 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 85P1B3 intrabodies in orderto achieve the desired targeting. Such 85P1B3 intrabodies are designedto bind specifically to a particular 85P1B3 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 85P1B3protein are used to prevent 85P1B3 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 85P1B3 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 85P1B3 with Recombinant Proteins

In another approach, recombinant molecules bind to 85P1B3 and therebyinhibit 85P1B3 function. For example, these recombinant moleculesprevent or inhibit 85P1B3 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 85P1B3specific antibody molecule. In a particular embodiment, the 85P1B3binding domain of a 85P1B3 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 85P1B3 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 85P1B3, whereby thedimeric fusion protein specifically binds to 85P1B3 and blocks 85P1B3interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of 85P1B3 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 85P1B3 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 85P1B3 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 85P1B3gene comprises contacting the 85P1B3 gene with a 85P1B3 antisensepolynucleotide. In another approach, a method of inhibiting 85P1B3 mRNAtranslation comprises contacting the 85P1B3 mRNA with an antisensepolynucleotide. In another approach, a 85P1B3 specific ribozyme is usedto cleave the 85P1B3 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 85P1B3 gene, such as the 85P1B3 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a85P1B3 gene transcription factor are used to inhibit 85P1B3 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 85P1B3 by interferingwith 85P1B3 transcriptional activation are also useful to treat cancersexpressing 85P1B3. Similarly, factors that interfere with 85P1B3processing are useful to treat cancers that express 85P1B3. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 85P1B3(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 85P1B3 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 85P1B3 antisensepolynucleotides, ribozymes, factors capable of interfering with 85P1B3transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 85P1B3 to a bindingpartner, etc.

In vivo, the effect of a 85P1B3 therapeutic composition can be evaluatedin a suitable animal model. For example, xenogenic prostate cancermodels can be used, wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice (Klein, et al., Nature Medicine (1997) 3:402-408).For example, PCT Patent Application WO 98/16628, Sawyers, et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. In vivo assays that evaluate the promotion ofapoptosis are useful in evaluating therapeutic compositions. In oneembodiment, xenografts from tumor bearing mice treated with thetherapeutic composition can be examined for the presence of apoptoticfoci and compared to untreated control xenograft-bearing mice. Theextent to which apoptotic foci are found in the tumors of the treatedmice provides an indication of the therapeutic efficacy of thecomposition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 85P1B3-related protein or a 85P1B3 gene ormessage, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 85P1B3 Gene

To isolate genes that are involved in the progression of androgendependent (AD) prostate cancer to androgen independent (AI) cancer, weconducted an experiment with the LAPC-4 AD xenograft in male SCID mice.Mice that harbored LAPC-4 AD xenografts were castrated when the tumorsreached a size of 1 cm in diameter. The tumors regressed in size andtemporarily stopped producing the androgen dependent protein PSA. Sevento fourteen days post-castration, PSA levels were detectable again inthe blood of the mice. Eventually the tumors develop an AI phenotype andstart growing again in the castrated males. Tumors were harvested atdifferent time points after castration to identify genes that are turnedon or off during the transition to androgen independence.

The gene 85P1B3 was derived from an LAPC-4 AD (3 days post-castration)minus LAPC-4 AD subtraction. The SSH DNA sequence of 319 bp (FIG. 1) isa fragment of the Opa-Interacting Protein 5 gene (OIP-5).

Materials and Methods

LAPC Xenografts and Human Tissues:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein, et al., Nature Med. (1997) 3:402-408;Craft, et al., Cancer Res. (1999) 59:5030-5036). Androgen dependent andindependent LAPC-4 AD and AI xenografts were grown in male SCID mice andwere passaged as small tissue chunks in recipient males. LAPC-4 AIxenografts were derived from LAPC-4 AD tumors, respectively. To generatethe AI xenografts, male mice bearing AD tumors were castrated andmaintained for 2-3 months. After the tumors re-grew, the tumors wereharvested and passaged in castrated males or in female SCID mice.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in TRIzol® reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA Synthesis Primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 714)

Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCC (SEQ ID NO: 715)GCCCGGGCAG3′ 3′GGCCCGTCCTAG5′ (SEQ ID NO: 716)

Adaptor 2: 5′GTAATACGACTCACTATAGGGCAGCGTGGTCG (SEQ ID NO: 717)CGGCCGAG3′ 3′CGGCTCCTAG5′ (SEQ ID NO: 718)

PCR Primer 1: 5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 719)

Nested Primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 720)

Nested Primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 721)

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts.Specifically, to isolate genes that are involved in the progression ofandrogen dependent (AD) prostate cancer to androgen independent (AI)cancer, an experiment was conducted with the LAPC-4 AD xenograft in maleSCID mice. Mice that harbored LAPC-4 AD xenografts were castrated whenthe tumors reached a size of 1 cm in diameter. The tumors regressed insize and temporarily stopped producing the androgen dependent proteinPSA. Seven to fourteen days post-castration, PSA levels were detectableagain in the blood of the mice. Eventually the tumors develop an AIphenotype and start growing again in the castrated males. Tumors wereharvested at different time points after castration to identify genesthat are turned on or off during the transition to androgenindependence.

The gene 85P1B3 was derived from an LAPC-4 AD (3 days post-castration)minus LAPC-4 AD subtraction. The SSH DNA sequence (FIG. 1) wasidentified.

The cDNA derived from an LAPC-4 AD tumor (grown in intact male mouse)was used as the source of the “driver” cDNA, while the cDNA from theLAPC-4 AD tumor (3 days post-castration) was used as the source of the“tester” cDNA. Double stranded cDNAs corresponding to tester and drivercDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from therelevant xenograft tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from the human cell lines HeLa, 293, A431,Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 mm.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:722) and 5′agccacacgcagctcattgtagaagg3′ (SEQ ID NO: 723) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTP's, 1×PCR buffer(Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3) and1×KlenTaq® DNA polymerase (Clontech). Five μl of the PCR reaction can beremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: Initial denaturation can be at 94° C.for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C.for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried outfor 2 min. After agarose gel electrophoresis, the band intensities ofthe 283 b.p. β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization can be required to achieveequal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 85P1B3 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

A typical RT-PCR expression analysis is shown in FIG. 10. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Strong expression of 85P1B3 wasobserved in xenograft pool, bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, breast cancer pool, ovary cancerpool, and cancer metastasis pool. Lower levels of expression wereobserved in VP1, VP2, and prostate cancer pool.

Example 2 Full Length Cloning of 85P1B3

To isolate genes that are involved in the progression of androgendependent (AD) prostate cancer to androgen independent (AI) cancer, anexperiment was conducted with the LAPC-4 AD xenograft in male SCID mice.Mice that harbored LAPC-4 AD xenografts were castrated when the tumorsreached a size of 1 cm in diameter. The tumors regressed in size andtemporarily stopped producing the androgen dependent protein PSA. Sevento fourteen days post-castration, PSA levels were detectable again inthe blood of the mice. Eventually the tumors develop an AI phenotype andstart growing again in the castrated males. Tumors were harvested atdifferent time points after castration to identify genes that are turnedon or off during the transition to androgen independence.

The gene 85P1B3 was derived from an LAPC-4 AD (3 days post-castrationminus LAPC-4 AD) (no castration) subtraction. The SSH DNA sequence(FIG. 1) was designated 85P1B3. cDNA clone 85P1B3-clone A (FIG. 2) wasidentified by screening a human testis library (Display Target, Pangene)using the 85P1B3 SSH DNA sequence. The cDNA (clone A) of 1,262 bprevealed an ORF encoding 229 amino acids (FIG. 2 and FIG. 3). Thenucleotide and protein sequence of 85P1B3 corresponds to the OIP-5 gene(FIG. 4). The 85P1B3 protein is predicted to be cytoplasmic using thePSORT program on the INTERNET.

Example 3 Chromosomal Localization

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available in the art,including fluorescent in situ hybridization (FISH), human/hamsterradiation hybrid (RH) panels (Walter, et al., Nature Genetics (1994)7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cellhybrid panels such as is available from the Coriell Institute (Camden,N.J.), and genomic viewers utilizing BLAST homologies to sequenced andmapped genomic clones (NCBI, Bethesda, Md.).

85P1B3 Maps to chromosome 15q14, using 85P1B3 sequence and the NCBIBLAST tool found on the INTERNET.

The chromosomal localization of 85P1B3 was also determined using theGeneBridge 4 Human/Hamster radiation hybrid (RH) panel (Walter, et al.,Nature Genetics (1994) 7:22) (Research Genetics, Huntsville Ala.).

The following PCR primers were used: 85P1B3.1 5′catgggactctgcatcttaattcc 3′ (SEQ ID NO: 732) 85P1B3.2 5′caggttcaggctttattgctgtct 3′ (SEQ ID NO: 733)

The resulting 85P1B3 mapping vector for the 93 radiation hybrid panelDNAs(100100010101000101000000000000110100000012101100001011100100001011100010010101100110110110101), and the mapping program available on the INTERNET,localize the 85P1B3 gene to chromosome 15q13.2-q14.

Of note, chromosome 15q13.2-q14 is a region implicated in cancers(Tomlinson, et al., Gastroenterology (1999) 116:789-795).

Example 4 Expression Analysis of 85P1B3 in Normal Tissues and PatientSpecimens

Analysis of 85P1B3 by RT-PCR is shown in FIG. 10. Strong expression of85P1B3 is observed in xenograft pool, bladder cancer pool, kidney cancerpool, colon cancer pool, lung cancer pool, breast cancer pool, ovarycancer pool, and cancer metastasis pool. Lower levels of expression areobserved in VP1, VP2, and prostate cancer pool.

Extensive Northern blot analysis of 85P1B3 in 16 human normal tissuesdemonstrated that 85P1B3 expression is reminiscent of a cancer-testisgene (FIG. 11). A 1.4 kb transcript was detected in testis but not inany other normal tissues. 85P1B3 expression was also shown in prostatecancer xenografts and in all cancer cell lines tested, such as in thecancers of the prostate (LAPC 4AD, LAPC 4AI, LAPC 9AD, LAPC 9AI, LNCaP,PC-3, DU145, Tsu-Prl and LAPC-4 CL), bladder (HT1197, SCaBER, UM-UC-3,TCCSUP, J82, 5637), 293T cell line, Ewing's sarcoma (EWS), brain(PFSK-1, T98G), bone (SK-ES-1, HOS, U-2 OS, RD-ES), lung (CALU-1, A427,NCI-H82, NCI-H146), kidney (769-P, A498, CAKI-1, SW839), breast (CAMA-1,DU4475, MCF-7, MDA-MB-435s), testicular (NTERRA-2, NCCIT, TERA-1,TERA-2), ovarian (OV-1063, PA-1, SW 626), pancreas (PANC-1, Bx PC-3,HPAC, Capan-1), colon (Caco-2, LoVo, T84, Colo205), and cervical (A431)(FIG. 12). These results indicate that 85P1B3 is a testis-specific genethat is upregulated in multiple cancers.

Expression of 85P1B3 was assayed in a panel of human patient tumors (T)and their respective matched normal tissues (N) on RNA dot blots (FIG.13). 85P1B3 expression was seen in the cancers of the breast, prostate,uterus, ovary, cervix, stomach and lung. The expression detected innormal adjacent tissues (isolated from diseased tissues) but not innormal tissues (isolated from healthy donors) may indicate that thesetissues are not fully normal and that 85P1B3 may be expressed in earlystage tumors. 85P1B3 was also found to be highly expressed in all humancancer cell lines tested, HeLa (cervical carcinoma), Daudi (Burkitt'slymphoma), K562 (CML), HL-60 (PML), G361 (melanoma), A549 (lungcarcinoma), MOLT-4 (lymphoblastic leukemia), SW480 (colorectalcarcinoma), and Raji (Burkitt's lymphoma).

Northern blot analysis on individual patient tumor specimens showedexpression of 85P1B3 in two colon tumor tissues tested, and in the coloncancer cell lines Colo 205, LoVo, T84 and Caco-2, but not in normalcolon (FIG. 14).

Expression of 85P1B3 was also detected in the tumors of 4 out of 5bladder cancer patients, and in all three bladder cancer cell linestested, but not in normal bladder (FIG. 15).

In lung cancer samples, 85P1B3 expression was observed in three lungtumor specimens, all three lung cancer cell lines tested, but not innormal lung (FIG. 16).

In order to assay for androgen regulation of 85P1B3 expression, LAPC-9ADtumor cells were injected in male mice (FIG. 17). When tumors reached apalpable size (0.3-0.5 cm in diameter), mice were castrated and tumorsharvested at different time points. RNA was isolated from the xenografttissues and Northern blots with 10 μg of total RNA/lane were probed withthe 85P1B3 SSH fragment. Results showed that expression of 85P1B3 is notaffected by androgen deprivation, and therefore, is notandrogen-regulated.

The restricted expression of 85P1B3 in normal tissues and the expressiondetected in bladder cancer, kidney cancer, colon cancer, lung cancer,prostate cancer, ovarian cancer, and breast cancer indicate that 85P1B3is a therapeutic and/or prophylactic target and a prognostic and/ordiagnostic marker for human cancers.

Example 5 Production of Recombinant 85P1B3 in Prokaryotic Systems

To express recombinant 85P1B3 in prokaryotic cells, the full or partiallength 85P1B3 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 85P1B3 are expressed in these constructs, amino acids 1 to229; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from85P1B3, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 85P1B3 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 85P1B3 cDNA. The pCRIIvector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 85P1B3 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 85P1B3 at the RNA level. Transcribed 85P1B3 RNArepresenting the cDNA amino acid coding region of the 85P1B3 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize85P1B3 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 85P1B3 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 85P1B3 cDNA protein coding sequence are fused to the GSTgene by cloning into pGEX-6P-1 or any other GST-fusion vector of thepGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). Theseconstructs allow controlled expression of recombinant 85P1B3 proteinsequences with GST fused at the amino-terminus and a six histidineepitope (6×His)(SEQ ID NO: 709) at the carboxyl-terminus. The GST and6×His (SEQ ID NO: 709) tags permit purification of the recombinantfusion protein from induced bacteria with the appropriate affinitymatrix and allow recognition of the fusion protein with anti-GST andanti-His antibodies. The 6×His tag (SEQ ID NO: 709) is generated byadding 6 histidine codons to the cloning primer at the 3′ end, e.g., ofthe open reading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 85P1B3-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

In one embodiment, a GST-fusion protein encoding the full length 85P1B3protein sequence (amino acids 1-229) was constructed and purified frominduced bacteria. This preparation was then used as immunogen togenerate a rabbit anti-85P1B3 polyclonal antibody (see the sectionentitled “Generation of 85P1B3 Polyclonal Antibodies”. As can be seen inFIG. 20A, the pAb strongly recognizes the original GST-fusion immunogenas well as 85P1B3 protein expressed in 293T cells (FIG. 20B and FIG.20C).

pMAL Constructs: To generate, in bacteria, recombinant 85P1B3 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the85P1B3 cDNA protein coding sequence are fused to the MBP gene by cloninginto the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly,Mass.). These constructs allow controlled expression of recombinant85P1B3 protein sequences with MBP fused at the amino-terminus and a6×His epitope tag (SEQ ID NO: 709) at the carboxyl-terminus. The MBP and6×His tags (SEQ ID NO: 709) permit purification of the recombinantprotein from induced bacteria with the appropriate affinity matrix andallow recognition of the fusion protein with anti-MBP and anti-Hisantibodies. The 6×His epitope tag (SEQ ID NO: 709) is generated byadding 6 histidine codons to the 3′ cloning primer. A Factor Xarecognition site permits cleavage of the pMAL tag from 85P1B3. ThepMAL-c2X and pMAL-p2X vectors are optimized to express the recombinantprotein in the cytoplasm or periplasm respectively. Periplasm expressionenhances folding of proteins with disulfide bonds.

pET Constructs: To express 85P1B3 in bacterial cells, all or parts ofthe 85P1B3 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 85P1B3 protein in bacteria with andwithout fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His (SEQ ID NO: 709) andS-Tag™ that aid purification and detection of the recombinant protein.For example, constructs are made utilizing pET NusA fusion system 43.1such that regions of the 85P1B3 protein are expressed as amino-terminalfusions to NusA.

C. Yeast Constructs:

pESC Constructs: To express 85P1B3 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 85P1B3 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 85P1B3. In addition, expression in yeastyields similar post-translational modifications, such as glycosylationsand phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 85P1B3 in the yeast species Saccharomycespombe, all or parts of the 85P1B3 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 85P1B3 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 6 Production of Recombinant 85P1B3 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 85P1B3 in eukaryotic cells, the full or partiallength 85P1B3 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 85P1B3 are expressed in these constructs, amino acids 1 to229; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from85P1B3, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-85P1B3 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 85P1B3 in mammalian cells, the85P1B3 ORF, or portions thereof, of 85P1B3 are cloned into pcDNA4/HisMaxVersion A (Invitrogen, Carlsbad, Calif.). Protein expression is drivenfrom the cytomegalovirus (CMV) promoter and the SP16 translationalenhancer. The recombinant protein has Xpress™ and six histidine (6×His)(SEQ ID NO: 709) epitopes fused to the amino-terminus. The pcDNA4/HisMaxvector also contains the bovine growth hormone (BGH) polyadenylationsignal and transcription termination sequence to enhance mRNA stabilityalong with the SV40 origin for episomal replication and simple vectorrescue in cell lines expressing the large T antigen. The Zeocin™resistance gene allows for selection of mammalian cells expressing theprotein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 85P1B3 in mammalian cells, the85P1B3 ORF, or portions thereof, of 85P1B3 with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope (SEQ ID NO: 709) fused to thecarboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovinegrowth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability, along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene can beused, as it allows for selection of mammalian cells expressing theprotein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 85P1B3 in mammalian cells andto allow detection of the recombinant proteins using fluorescence, the85P1B3 ORF, or portions thereof, of 85P1B3 with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, CA). Protein expression is driven from the cytomegalovirus(CMV) promoter. The recombinant proteins have the Green FluorescentProtein (GFP) fused to the carboxyl-terminus facilitating non-invasive,in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPOvector also contains the bovine growth hormone (BGH) polyadenylationsignal and transcription termination sequence to enhance mRNA stabilityalong with the SV40 origin for episomal replication and simple vectorrescue in cell lines expressing the large T antigen. The Neomycinresistance gene allows for selection of mammalian cells that express theprotein, and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of the 85P1B3 proteins.

PAPtag: The 85P1B3 ORF, or portions thereof, of 85P1B3 are cloned intopAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates analkaline phosphatase fusion at the carboxyl-terminus of the 85P1B3proteins while fusing the IgGK signal sequence to the amino-terminus.Constructs are also generated in which alkaline phosphatase with anamino-terminal IgGκ signal sequence is fused to the amino-terminus of85P1B3 proteins. The resulting recombinant 85P1B3 proteins are optimizedfor secretion into the media of transfected mammalian cells and can beused to identify proteins such as ligands or receptors that interactwith the 85P1B3 proteins. Protein expression is driven from the CMVpromoter and the recombinant proteins also contain myc and 6×Hisepitopes (SEQ ID NO: 709) fused at the carboxyl-terminus thatfacilitates detection and purification. The Zeocin resistance genepresent in the vector allows for selection of mammalian cells expressingthe recombinant protein and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

ptag5: The 85P1B3 ORF, or portions thereof, of 85P1B3 was cloned intopTag-5. This vector is similar to pAPtag but without the alkalinephosphatase fusion. This construct generated 85P1B3 protein with anamino-terminal IgGK signal sequence and myc and 6×His epitope (SEQ IDNO: 709) tags at the carboxyl-terminus that facilitate detection andaffinity purification. The resulting recombinant 85P1B3 protein wasoptimized for secretion into the media of transfected mammalian cells,and was used as immunogen or ligand to identify proteins such as ligandsor receptors that interact with the 85P1B3 proteins. Protein expressionis driven from the CMV promoter. The Zeocin resistance gene present inthe vector allows for selection of mammalian cells expressing theprotein, and the ampicillin resistance gene permits selection of theplasmid in E. coli.

PsecFc: The 85P1B3 ORF, or portions thereof, of 85P1B3 are also clonedinto psecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, California). This construct generates an IgG1 Fc fusion atthe carboxyl-terminus of the 85P1B3 proteins, while fusing the IgGKsignal sequence to N-terminus. 85P1B3 fusions utilizing the murine IgG1Fc region are also used. The resulting recombinant 85P1B3 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with the 85P1B3 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 85P1B3,or portions thereof, constitutively, the ORF of 85P1B3 was cloned intopSRα constructs. Amphotropic and ecotropic retroviruses were generatedby transfection of pSRα constructs into the 293T-10A1 packaging line orco-transfection of pSRα and a helper plasmid (containing deletedpackaging sequences) into the 293 cells, respectively. The retroviruswas used to infect a variety of mammalian cell lines, resulting in theintegration of the cloned gene, 85P1B3, into the host cell-lines.Protein expression is driven from a long terminal repeat (LTR). TheNeomycin resistance gene present in the vector allows for selection ofmammalian cells that express the protein, and the ampicillin resistancegene and ColE1 origin permit selection and maintenance of the plasmid inE. coli. FIG. 18 shows expression of 85P1B3 using the pSRα retroviralvector in the prostate cancer cell line PC3. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, SCaBER, NIH-3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 85P1B3 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 734) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×His (SEQID NO: 709) fusion proteins of the full-length 85P1B3 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 85P1B3. High virus titerleading to high level expression of 85P1B3 is achieved in viral deliverysystems such as adenoviral vectors and herpes amplicon vectors. The85P1B3 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy™ shuttle vector (Stratagene). Recombinationand virus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 85P1B3coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as SCaBER,NIH-3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 85P1B3 inmammalian cells, coding sequences of 85P1B3, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant85P1B3. These vectors are thereafter used to control expression of85P1B3 in various cell lines such as SCaBER, NIH-3T3, 293 or rat-1cells.

B. Baculovirus Expression Systems

To generate recombinant 85P1B3 proteins in a baculovirus expressionsystem, 85P1B3 ORF, or portions thereof, are cloned into the baculovirustransfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag atthe N-terminus. Specifically, pBlueBac-85P1B3 is co-transfected withhelper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda)insect cells to generate recombinant baculovirus (see Invitrogeninstruction manual for details). Baculovirus is then collected from cellsupernatant and purified by plaque assay.

Recombinant 85P1B3 protein is then generated by infection of HighFive™insect cells (Invitrogen) with purified baculovirus. Recombinant 85P1B3protein can be detected using anti-85P1B3 or anti-His-tag antibody.85P1B3 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 85P1B3.

Example 7 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 85P1B3 amino acid sequence, each assessmentavailable by accessing the ProtScale website on the ExPasy molecularbiology server on the INTERNET.

These profiles: FIG. 5, Hydrophilicity, (Hopp, T. P., et al., Proc.Natl. Acad. Sci. U.S.A. (1981) 78:3824-3828); FIG. 6, Hydropathicity,(Kyte, J., et al., J. Mol. Biol. (1982) 157:105-132); FIG. 7, PercentageAccessible Residues (Janin, J., Nature (1979) 277:491-492); FIG. 8,Average Flexibility, (Bhaskaran, R., et al., Int. J. Pept. Protein Res.(1988) 32:242-255); FIG. 9, Beta-turn (Deleage, G., et al., ProteinEngineering (1987) 1:289-294); and optionally others available in theart, such as on the ProtScale website, were used to identify antigenicregions of the 85P1B3 protein. Each of the above amino acid profiles of85P1B3 were generated using the following ProtScale parameters foranalysis: 1) A window size of 9; 2) 100% weight of the window edgescompared to the window center; and, 3) amino acid profile valuesnormalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 85P1B3 protein indicated, e.g., by theprofiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 areused to prepare immunogens, either peptides or nucleic acids that encodethem, to generate therapeutic and diagnostic anti-85P1B3 antibodies. Theimmunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 85P1B3 protein. In particular, peptide immunogens of theinvention can comprise, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 229 that includes an aminoacid position having a value greater than 0.5 in the Hydrophilicityprofile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 229 that includes an amino acidposition having a value less than 0.5 in the Hydropathicity profile ofFIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in anywhole number increment up to 229 that includes an amino acid positionhaving a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 229 that includes an amino acidposition having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8; and, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 229 that includes an aminoacid position having a value greater than 0.5 in the Beta-turn profileof FIG. 9. Peptide immunogens of the invention can also comprise nucleicacids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 85P1B3, namely the predicted presence andlocation of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence using theHNN—Hierarchical Neural Network method (Guermeur, 1997), accessed fromthe ExPasy molecular biology server on the INTERNET. The analysisindicates that 85P1B3 is composed of 36.8% alpha helix, 13.97% extendedstrand, and 49.34% random coil (FIG. 21A).

Analysis for the potential presence of transmembrane domains in 85P1B3was carried out using a variety of transmembrane prediction algorithmsaccessed from the ExPasy molecular biology server on the INTERNET. Apotential transmembrane domain composed of amino acids 129-149 ispredicted by the TMpred program (FIG. 21B). HMMTop predicts atransmembrane region from amino acids 134-158. The SOSUI and TMHMM (FIG.21C) programs predict that 85P1B3 is a soluble protein withouttransmembrane domains. The results of the transmembrane predictions aresummarized in Table XXV.

Example 8 Generation of 85P1B3 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 85P1B3 protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 85P1B3).

For example, 85P1B3 recombinant bacterial fusion proteins or peptidesencoding hydrophilic, flexible, beta-turn regions of the 85P1B3sequence, such as amino acids 1-77 and 190-229 are used as antigens togenerate polyclonal antibodies in New Zealand White rabbits. It isuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include, but are not limited to, keyhole limpet hemocyanin(KLH), serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. In one embodiment, a peptide encoding amino acids 190-206 of85P1B3 is conjugated to KLH and used to immunize the rabbit.Alternatively the immunizing agent may include all or portions of the85P1B3 protein, analogs or fusion proteins thereof. For example, the85P1B3 amino acid sequence can be fused using recombinant DNA techniquesto any one of a variety of fusion protein partners that are well knownin the art, such as glutathione-5-transferase (GST) and HIS taggedfusion proteins. Such fusion proteins are purified from induced bacteriausing the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding the full length 85P1B3protein sequence was produced and purified and used as immunogen (seethe section entitled “Production of 85P1B3 in Prokaryotic Systems”).Shorter sequences are also fused to GST in order to direct antibody tospecific regions of the protein such as amino acids 1-77 to generateamino-terminal specific antibodies. Other recombinant bacterial fusionproteins that may be employed include maltose binding protein, LacZ,thioredoxin, NusA, or an immunoglobulin constant region (see the sectionentitled “Production of 85P1B3 in Prokaryotic Systems” and CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul,et al., eds., 1995; Linsley, P. S., et al., J. Exp. Med. (1991)174:561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 85P1B3 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, a predictedantigenic region of 85P1B3, amino acids 190-229, is cloned into the Tag5mammalian secretion vector. The recombinant protein is purified by metalchelate chromatography from tissue culture supernatants of 293T cellsstably expressing the recombinant vector. The purified Tag5 85P1B3protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum raised from immunization with GST-85P1B3 full length fusionprotein, the full-length 85P1B3 cDNA was cloned into pcDNA 3.1 myc-hisexpression vector (Invitrogen, see the Example entitled “Production ofRecombinant 85P1B3 in Eukaryotic Systems”). After transfection of theconstructs into 293T cells, cell lysates were probed with theanti-85P1B3 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 85P1B3 protein using the Western blot technique. As can beseen in FIG. 20B, the anti-85P1B3 pAb specifically recognized 85P1B3protein expressed in 293T cells that is the same molecular weight asthat detected by the anti-His Ab (FIG. 20C). Recognition of nativeprotein by the antiserum is determined by immunoprecipitation and flowcytometric analyses of 293T and other recombinant 85P1B3-expressingcells. In addition, specificity of the antiserum is tested by Westernblot, immunoprecipitation, fluorescent microscopy, and flow cytometrictechniques using cells that endogenously express 85P1B3.

To purify the anti-serum derived from the GST-85P1B3 immunized rabbit,the serum was passed over an affinity column composed of GST to removeanti-GST reactive antibodies. The serum was then further purified byprotein G affinity chromatography to isolate the IgG fraction. Serumfrom rabbits immunized with other fusion proteins, such as MBP fusionproteins, are purified by depletion of antibodies reactive to MBP, orother fusion partner sequence, by passage over an affinity columncontaining the fusion partner either alone or in the context of anirrelevant fusion protein. Sera from His-tagged protein and peptideimmunized rabbits as well as fusion partner depleted sera are furtherpurified by passage over an affinity column composed of the originalprotein immunogen or free peptide coupled to Affigel matrix (BioRad).

Example 9 Generation of 85P1B3 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 85P1B3 comprise those that reactwith epitopes of the protein that would disrupt or modulate thebiological function of 85P1B3, for example those that would disrupt itsinteraction with ligands or proteins that mediate or are involved in itsbiological activity. Therapeutic mAbs also comprise those whichspecifically bind epitopes of 85P1B3 exposed on the cell surface andthus are useful in targeting mAb-toxin conjugates. Immunogens forgeneration of such mAbs include those designed to encode or contain theentire 85P1B3 protein or regions of the 85P1B3 protein predicted to beantigenic from computer analysis of the amino acid sequence (see, e.g.,FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled“Antigenicity Profiles”).

Immunogens include peptides, recombinant bacterial proteins, andmammalian expressed Tag 5 proteins and human and murine IgG FC fusionproteins. To generate mAbs to 85P1B3, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogenmixed in complete Freund's adjuvant. Mice are then subsequentlyimmunized IP every 2-4 weeks with, typically, 10-50 μg of antigen mixedin incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is usedin immunizations. In addition, a DNA-based immunization protocol isemployed in which a mammalian expression vector encoding 85P1B3 sequenceis used to immunize mice by direct injection of the plasmid DNA. Forexample, either pcDNA 3.1 encoding the full length 85P1B3 cDNA, aminoacids 1-77, or 190-229 of 85P1B3 (predicted to be antigenic fromsequence analysis, see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8 or FIG. 9)fused at the amino-terminus to an IgK leader sequence and at thecarboxyl-terminus to the coding sequence of murine or human IgG Fcregion, is used. This protocol is used alone and in combination withprotein immunogens. Test bleeds are taken 7-10 days followingimmunization to monitor titer and specificity of the immune response.Once appropriate reactivity and specificity is obtained as determined byELISA, Western blotting, immunoprecipitation, fluorescence microscopy,and flow cytometric analyses, fusion and hybridoma generation is thencarried out with established procedures well known in the art (see,e.g., Harlow and Lane, 1988).

In one embodiment for generating 85P1B3 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encoding the full length85P1B3 protein is expressed and purified. A cleavage fragment encoding85P1B3 specific amino acids is then used as immunogen in which GST isremoved by site-specific proteolysis. Balb C mice are initiallyimmunized intraperitoneally with 25 μg of the 85P1B3 cleavage proteinmixed in complete Freund's adjuvant. Mice are subsequently immunizedevery two weeks with 25 μg of 85P1B3 cleavage protein mixed inincomplete Freund's adjuvant for a total of three immunizations. Thetiter of serum from immunized mice is determined by ELISA using the fulllength GST-fusion protein and the cleaved immunogen. Reactivity andspecificity of serum to full length 85P1B3 protein is monitored byWestern blotting, immunoprecipitation and flow cytometry using 293Tcells transfected with an expression vector encoding the 85P1B3 cDNA.Other recombinant 85P1B3-expressing cells (see e.g., the Exampleentitled “Production of 85P1B3 in Eukaryotic Systems”) or cellsendogenously expressing 85P1B3 are also used. Mice showing the strongestreactivity are rested and given a final injection of 85P1B3 cleavageprotein in PBS and then sacrificed four days later. The spleens of thesacrificed mice are harvested and fused to SPO/2 myeloma cells usingstandard procedures (Harlow and Lane, 1988). Supernatants from growthwells following HAT selection are screened by ELISA, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometry toidentify 85P1B3 specific antibody-producing clones.

The binding affinity of a 85P1B3 monoclonal antibody is determined usingstandard technologies. Affinity measurements quantify the strength ofantibody to epitope binding and are used to help define which 85P1B3monoclonal antibodies preferred for diagnostic or therapeutic use, asappreciated by one of skill in the art. The Biacore™ system (Uppsala,Sweden) is a preferred method for determining binding affinity. TheBiacore™ system uses surface plasmon resonance (SPR, Welford, K., Opt.Quant. Elect. (1991) 23:1; Morton, et al., Methods in Enzymology (1998)295:268) to monitor biomolecular interactions in real time. BIAcoreanalysis conveniently generates association rate constants, dissociationrate constants, equilibrium dissociation constants, and affinityconstants.

Example 10 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney, et al., Current Protocols inImmunology (1998) 18.3.1; Sidney, et al., J. Immunol. (1995) 154:247;Sette, et al., Mol. Immunol. (1994) 31:813). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides.

Example 11 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVIIIemploy the protein sequence data from the gene product of 85P1B3 setforth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 85P1B3protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

-   -   “ΔG”=a_(1i)×a_(2i)×a_(3i) . . . ×a_(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (i) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota, et al., J. Mol. Biol. (1997) 267:1258-1267; (seealso Sidney, et al., Human Immunol. (1996) 45:79-93; and Southwood, etal., J. Immunol. (1998) 160:3363-3373). Briefly, for all i positions,anchor and non-anchor alike, the geometric mean of the average relativebinding (ARB) of all peptides carrying j is calculated relative to theremainder of the group, and used as the estimate of j_(i). For Class IIpeptides, if multiple alignments are possible, only the highest scoringalignment is utilized, following an iterative procedure. To calculate analgorithm score of a given peptide in a test set, the ARB valuescorresponding to the sequence of the peptide are multiplied. If thisproduct exceeds a chosen threshold, the peptide is predicted to bind.Appropriate thresholds are chosen as a function of the degree ofstringency of prediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Complete protein sequences from 85P1B3 are scanned utilizing motifidentification software, to identify 8-, 9- 10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 85P1B3 protein sequence scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 85P1B3 protein is also analyzed for the presence of 8-, 9- 10-, or11-mer peptides with the HLA-B7-supermotif. Corresponding peptides aresynthesized and tested for binding to HLA-B*0702, the molecule encodedby the most common B7-supertype allele (i.e., the prototype B7 supertypeallele). Peptides binding B*0702 with IC₅₀ of <500 nM are identifiedusing standard methods. These peptides are then tested for binding toother common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, andB*5401). Peptides capable of binding to three or more of the fiveB7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the85P1B3 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 12 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTL's to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMC's are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to theDC's on day 6 at 75 ng/ml and the cells are used for CTL inductioncultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMC's are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe non-adherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare re-stimulated with peptide-pulsed adherent cells. The PBMC's arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMC's are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the non-adherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml 12 microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai, et al., Critical Reviews in Immunology (1998)18:65-75). Seven days later, the cultures are assayed for CTL activityin a ⁵¹Cr release assay. In some experiments the cultures are assayedfor peptide-specific recognition in the in situ IFNγ ELISA at the timeof the second restimulation followed by assay of endogenous recognition7 days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Trition X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTL's (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV—transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 110% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTL's in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMC's isolated from patientsbearing a tumor that expresses 85P1B3. Briefly, PBMC's are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24, etc.,are also confirmed using similar methodology.

Example 13 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g., greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analogued to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5,000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst, et al., J. Immunol. (1996) 157:2539; and Pogue, et al., Proc.Natl. Acad. Sci. USA (1995) 92:8166).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTL's are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney, et al. (J. Immunol. (1996)157:3480-3490).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTL's are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analogued peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with85P1B3-expressing tumors.

Other Analoguing Strategies

Another form of peptide analoguing, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcross-binding capabilities in some instances (see, e.g., the review bySette, et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 14 Identification and Confirmation of 85P1B3-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 85P1B3-derived, HLA class II HTL epitopes, the 85P1B3antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood, et al., J. Immunol. (1998) 160:3363-3373). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood, et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 85P1B3-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 85P1B3-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 85P1B3 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk, et al. (J. Immunol. (1994) 152:5742-5748). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 15 Immunogenicity of 85P1B3-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 85P1B3-expressing tumors.

Example 16 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney, et al., Human Immunol.(1996) 45:79-93). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

Immunogenicity studies in humans (e.g., Bertoni, et al., J. Clin.Invest. (1997) 100:503; Doolan, et al., Immunity (1997) 7:97; andThrelkeld, et al., J. Immunol. (1997) 159:1648) have shown that highlycross-reactive binding peptides are almost always recognized asepitopes. The use of highly cross-reactive binding peptides is animportant selection criterion in identifying candidate epitopes forinclusion in a vaccine that is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A., A Course In Game Theory MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 17 CTL Recognition of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 85P1B3 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 85P1B3 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 18 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTL's and HTL's in transgenicmice, by use of a 85P1B3-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 85P1B3-expressing tumor. The peptidecomposition can comprise multiple CTL and/or HTL epitopes. The epitopesare identified using methodology as described herein. This example alsoillustrates that enhanced immunogenicity can be achieved by inclusion ofone or more HTL epitopes in a CTL vaccine composition; such a peptidecomposition can comprise an HTL epitope conjugated to a CTL epitope. TheCTL epitope can be one that binds to multiple HLA family members at anaffinity of 500 nM or less, or analogs of that epitope. The peptides maybe lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander, et al., J. Immunol. (1997) 159:4753-4761). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are re-stimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello, et al., J. Exp. Med. (1991) 173:1007).

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity”. Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 19 Selection of CTL and HTL Epitopes for Inclusion in an85P1B3-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 85P1B3 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 85P1B3. Forexample, if it has been observed that patients who spontaneously clear85P1B3 generate an immune response to at least three (3) from 85P1B3antigen, then three or four (3-4) epitopes should be included for HLAclass I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, on the INTERNET.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 85P1B3, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 85P1B3.

Example 20 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 85P1B3, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 85P1B3 to providebroad population coverage, i.e., both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multi-epitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH₄)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts,et al., J. Immunol. (1996) 156:683-692; Demotz, et al., Nature (1989)342:682-684); or the number of peptide-HLA class I complexes can beestimated by measuring the amount of lysis or lymphokine release inducedby diseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama, et al., J. Immunol. (1995)154:567-576).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander, et al., Immunity (1994)1:751-761.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTL's invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTL's induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTL's in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTL's induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e., HTL's, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander,et al. Immunity (1994) 1:751-761). The results indicate the magnitude ofthe HTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett, et al., Aids Res. and HumanRetroviruses (1998) 14S3:S299-S309) or recombinant vaccinia, forexample, expressing a minigene or DNA encoding the complete protein ofinterest (see, e.g., Hanke, et al., Vaccine (1998) 16:439-445; Sedegah,et al., Proc. Natl. Acad. Sci USA (1998) 95:7648-1653; Hanke, et al.,Immunol. Letters (1999) 66:177-181; and Robinson, et al., Nature Med.(1999) 5:526-534).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 22 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent85P1B3 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 85P1B3-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freund's Adjuvant. The dose of peptide for theinitial immunization is from about 1 to about 50,000 μg, generally100-5,000 μg, for a 70 kg patient. The initial administration of vaccineis followed by booster dosages at 4 weeks followed by evaluation of themagnitude of the immune response in the patient, by techniques thatdetermine the presence of epitope-specific CTL populations in a PBMCsample. Additional booster doses are administered as required. Thecomposition is found to be both safe and efficacious as a prophylaxisagainst 85P1B3-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 23 Polyepitopic Vaccine Compositions Derived from Native 85P1B3Sequences

A native 85P1B3 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes is selected; it can be used to generate a minigene construct.The construct is engineered to express the peptide, which corresponds tothe native protein sequence. The “relatively short” peptide is generallyless than 250 amino acids in length, often less than 100 amino acids inlength, preferably less than 75 amino acids in length, and morepreferably less than 50 amino acids in length. The protein sequence ofthe vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. As noted herein, epitope motifs may be nestedor overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9-mer epitopes and one 10-merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 85P1B3 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 85P1B3, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 24 Polyepitopic Vaccine Compositions from Multiple Antigens

The 85P1B3 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 85P1B3 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 85P1B3 as well astumor-associated antigens that are often expressed with a target cancerassociated with 85P1B3 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 25 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 85P1B3. Suchan analysis can be performed in a manner described by Ogg, et al.,Science (1998) 279:2103-2106. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 85P1B3 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising an 85P1B3 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey, et al., N.Engl. J. Med. (1997) 337:1267). Briefly, purified HLA heavy chain(A*0201 in this example) and β2-microglobulin are synthesized by meansof a prokaryotic expression system. The heavy chain is modified bydeletion of the transmembrane-cytosolic tail and COOH-terminal additionof a sequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMC's are centrifuged at 300 g for 5 minutes and resuspended in 50 μlof cold phosphate-buffered saline. Tricolor analysis is performed withthe tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38.The PBMC's are incubated with tetramer and antibodies on ice for 30 to60 min and then washed twice before formaldehyde fixation. Gates areapplied to contain >99.98% of control samples. Controls for thetetramers include both A*0201-negative individuals and A*0201-positivenon-diseased donors. The percentage of cells stained with the tetrameris then determined by flow cytometry. The results indicate the number ofcells in the PBMC sample that contain epitope-restricted CTL's, therebyreadily indicating the extent of immune response to the 85P1B3 epitope,and thus the status of exposure to 85P1B3, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 26 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from85P1B3-associated disease or who have been vaccinated with an 85P1B3vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 85P1B3 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 ul of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andre-stimulated with peptide, rIL-2 and 105 irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. (1996) 2:1104, 1108;Rehermann, et al., J. Clin. Invest. (1996) 97:1655-1665; and Rehermann,et al., J. Clin. Invest. (1996) 98:1432-1440).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al., J.Virol. (1992) 66:2670-2678).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 85P1B3 or an 85P1B3 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 85P1B3 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 27 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 28 Phase II Trials in Patients Expressing 85P1B3

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses85P1B3. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTL's in cancer patients that express85P1B3, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTL's improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 85P1B3.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 85P1B3-associated disease.

Example 29 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against85P1B3 is generated.

Example 30 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APC's, or “professional” APC's such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 85P1B3 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. (1998) 4:328; Nature Med. (1996) 2:52 andProstate (1997) 32:272). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 85P1B3 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 31 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 85P1B3. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo, et al., J.Immunol. (1994) 152:3913). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 85P1B3 to isolate peptidescorresponding to 85P1B3 that have been presented on the cell surface.Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 32 Complementary Polynucleotides

Sequences complementary to the 85P1B3-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 85P1B3. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 85P1B3. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to the 85P1B3-encodingtranscript.

Example 33 Purification of Naturally-Occurring or Recombinant 85P1B3Using 85P1B3 Specific Antibodies

Naturally occurring or recombinant 85P1B3 is substantially purified byimmunoaffinity chromatography using antibodies specific for 85P1B3. Animmunoaffinity column is constructed by covalently coupling anti-85P1B3antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 85P1B3 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 85P1B3 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/85P1B3 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and GCRPis collected.

Example 34 Identification of Molecules which Interact with 85P1B3

85P1B3, or biologically active fragments thereof, are labeled with 121 lBolton-Hunter reagent. (See, e.g., Bolton, et al., Biochem. J. (1973)133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 85P1B3, washed, and anywells with labeled 85P1B3 complex are assayed. Data obtained usingdifferent concentrations of 85P1B3 are used to calculate values for thenumber, affinity, and association of 85P1B3 with the candidatemolecules. Throughout this application, various website data content,publications, applications and patents are referenced and re located onthe World Wide Web. The disclosures of each of these items ofinformation are hereby incorporated by reference herein in theirentireties.

Example 35 In Vivo Assay for 85P1B3 Tumor Growth Promotion

The effect of the 85P1B3 protein on tumor cell growth can be confirmedin vivo by gene overexpression in a variety of cancer cells, includingprostate, kidney and bladder. For example, SCID mice can be injected SQon each flank with 1×10⁶ prostate, kidney or bladder cancer cells (suchas PC3, LNCaP, SCaBER, UM-UC-3, HT1376, RT4, T24, Caki, A-498 and SW839cells) containing tkNeo empty vector or 85P1B3.

At least two strategies may be used: (1) Constitutive 85P1B3 expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems. (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., can be usedprovided such promoters are compatible with the host cell systems. Tumorvolume is then monitored at the appearance of palpable tumors and isfollowed over time to validate that 85P1B3-expressing cells grow at afaster rate and that tumors produced by 85P1B3-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice can be implanted with the same cellsorthotopically in the prostate, bladder or kidney to determine if 85P1B3has an effect on local growth in the prostate, bladder or kidney or onthe ability of the cells to metastasize, specifically to lungs or lymphnodes (Fu, X., et al., Int. J. Cancer (1991) 49:938-939; Chang, S., etal., Anticancer Res. (1997) 17:3239-3242; Peralta, E. A., et al., JUrol. (1999) 162:1806-1811).

Furthermore, this assay is useful to confirm the 85P1B3 inhibitoryeffect of candidate therapeutic compositions, such as for example,85P1B3 antibodies or intrabodies, and 85P1B3 antisense molecules orribozymes.

Example 36 85P1B3 Monoclonal Antibody-Mediated Inhibition of Tumors InVivo

The significant expression of 85P1B3 in cancer tissues, together withits restricted expression in normal tissues, makes 85P1B3 an excellenttarget for antibody therapy. In cases where the monoclonal antibodytarget is a cell surface protein, antibodies have been shown to beefficacious at inhibiting tumor growth (See, e.g., Saffran, D., et al.,PNAS (2001) 10:1073-1078). In cases where the target is not on the cellsurface, such as PSA and PAP in prostate cancer, antibodies have alsobeen shown to recognize and inhibit growth of cells expressing thoseproteins (Saffran, D. C., et al., Cancer and Metastasis Reviews (1999)18:437-449). As with any cellular protein with a restricted expressionprofile, 85P1B3 is a target for T cell-based immunotherapy.

Accordingly, the therapeutic efficacy of anti-85P1B3 mAbs in humancolon, kidney, bladder and prostate cancer mouse models is modeled in85P1B3-expressing kidney, bladder or prostate cancer xenografts orcancer cell lines, such as those described in the Example entitled “InVivo Assay for 85P1B3 Tumor Growth Promotion”, that have been engineeredto express 85P1B3.

Antibody efficacy on tumor growth and metastasis formation is confirmed,e.g., in a mouse orthotopic in the prostate, colon, bladder or kidneycancer xenograft model. The antibodies can be unconjugated, or can beconjugated to a therapeutic modality, as appreciated in the art. It isconfirmed that anti-85P1B3 mAbs inhibit formation of 85P1B3-expressingkidney, bladder and prostate tumors. Anti-85P1B3 mAbs also retard thegrowth of established orthotopic tumors and prolong survival oftumor-bearing mice. These results indicate the utility of anti-85P1B3mAbs in the treatment of local and advanced stages of cancer. (See,e.g., Saffran, D., et al., PNAS (2001) 10:1073-1078)

Administration of anti-85P1B3 mAbs retard established orthotopic tumorgrowth and inhibit metastasis to distant sites, resulting in asignificant prolongation in the survival of tumor-bearing mice. Thesestudies indicate that 85P1B3 is an attractive target for immunotherapyand demonstrate the therapeutic potential of anti-85P1B3 mAbs for thetreatment of local and metastatic kidney, colon, bladder and prostatecancer.

This example demonstrates that unconjugated 85P1B3 monoclonal antibodieseffectively to inhibit the growth of human bladder tumors grown in SCIDmice; accordingly a combination of such efficacious monoclonalantibodies is also effective.

Example 37 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 38 Splice Variants of 85P1B3

Splice variants are also called alternative transcripts. When a gene istranscribed from genomic DNA, the initial RNA is generally spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternatively spliced mRNA products. Alternativetranscripts each have a unique exon makeup, and can have differentcoding and/or non-coding (5′ or 3′ end) portions, from the originaltranscript. Alternative transcripts can code for similar proteins withthe same or a similar function or may encode proteins with differentfunctions, and may be expressed in the same tissue at the same time, orat different tissue at different times, proteins encoded by alternativetranscripts can have similar or different cellular or extracellularlocalizations, e.g., be secreted.

Splice variants are identified by a variety of art-accepted methods. Forexample, splice variants are identified by use of EST data. First, allhuman EST's were grouped into clusters which show direct or indirectidentity with each other. Second, EST's in the same cluster were furthergrouped into sub-clusters and assembled into a consensus sequence. Thestarting gene is compared to the consensus sequence(s). Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifysplice variants based on genomic sequences. Genomic-based variantidentification programs include FgenesH (Salamov, A., et al., GenomeResearch (2000) 10:516-522); Grail and GenScan as found on the INTERNET.For a general discussion of splice variant identification protocols see,e.g., Southan, C., FEBS Lett. (2001) 498:214-218; de Souza, S. J., etal., Proc. Natl. Acad Sci USA (2000) 97:12690-12693.

For variants identified by the EST-based method, Table XXI shows thenucleotide sequences of the splice variants. Figure Table XXII shows thealignment of the splice variant with the 85P1B3 nucleic acid sequence.Table XXIII displays the single longest alignment of an amino acidsequence encoded by a splice variant, out of all six potential readingframes with 85P1B3. Thus, for each splice variant, a variant's readingframe that encodes the longest single contiguous peptide homologybetween 85P1B3 and the variant is the proper reading frame orientationfor the variant. Due to the possibility of sequencing errors in EST orgenomic data, other peptides in the relevant reading frame orientation(5′ to 3′ or 3′ to 5′) can also be encoded by the variant. Table XXIVlays out all three frame shifted amino acid translations of the splicevariant for the identified reading frame orientation.

For variants identified by any one of the genomic sequence-basedmethods, Table XXI shows the nucleotide sequences of the splice variant.Figure Table XXII shows the alignment of the splice variant with the85P1B3 nucleic acid sequence. Table XXIII displays the alignment ofamino acid sequence of the predicted transcripts with 85P1B3. Thegenomic-based computer programs predict a transcript from genomicsequence, and not only predict exons but also set open reading frame asthe first forward open reading frame. The predicted transcript does notcontain 5′ or 3′ untranslated region (UTR). It starts with ATG and endswith a stop codon, TAG, TGA or TAA. In case the transcript is predictedon the reverse strand of the genomic sequence, the sequence of thetranscript is reverse-complemented to the genomic sequence of the exons.Thus, the genomic-based programs provide the correct transcriptsequence, with 5′ to 3′ orientation and +1 as the open reading frame.However, due to the possibility of inaccurate prediction of exons or thepossibility of sequencing errors in genomic data, other peptides inother forward open reading frame can also be encoded by the variant.Table XXIV lays out all amino acid translations of the splice variant ineach of the three forward reading frames.

To further confirm the parameters of a splice variant, a variety oftechniques are available in the art, such as proteomic validation,PCR-based validation, and 5′ RACE validation, etc. (see, e.g., ProteomicValidation: Brennan, S. O., et al., Biochim Biophys Acta. (1999)1433:321-326; Ferranti, P., et al., Eur J. Biochem. (1997) 249:1-7;PCR-based Validation: Wellmann, S., et al., Clin Chem. (2001)47:654-660; Jia, H. P., et al., Gene. (2001) 263:211-218; PCR-based and5′ RACE Validation: Brigle, K. E., et al., Biochim Biophys Acta. (1997)1353:191-198.

It is known in the art that genomic regions are upregulated in cancers.When the genomic region to which 85P1B3 maps is upregulated in aparticular cancer, the splice variants of 85P1B3 are upregulated aswell. Disclosed herein is that 85P1B3 has a particular expressionprofile. Splice variants of 85P1B3 that are structurally and/orfunctionally similar to 85P1B3 share this expression pattern, thusserving as tumor-associated markers/antigens.

Using the EST assembly approach, we identified one splice variantsdesignated splice variant 1. TABLE XXIA Nucleotide sequence of splicevariant 1 (SEQ ID NO: 701). 1 TTTTTTTTTT CCTATCTAGC TATCTCTTAAAAACAAAAGC CATAGTAAAT GCATCAGAGA 61 TGGATATTCA AAATGTTCCT CTATCAGAAAAGATTGCAGA GGTAAAATTT CATGATGGTT 121 GTATGCTTTT TTAAAATACA GACAACTCTTGATAACTTCT ACCAATGAAC TTGGGGATGA 181 TGAAATGGCA TGATGCTCAA TAATCCTTTTTACTTGATTT GACCTTCCCT ATTGAATTTG 241 TAATGAAAAA CAAAATACTA AAACCACACTGTAAGGTATA GTTCAGGAAG AAAGGAAAAG 301 CTGCTCAACT GCTGCACTCC TGCATTCTCCTTTGTGCTGG GAATGGATAT CATCATCTTG 361 CCATAGAGGT GTCTTCTTTG CAAATACCTTGTAATTGCTC AACTGTCTCA GACATAAGAG 421 TGATGAAACA GTTATTAAGA ATTCCTGGCCGGGCGTGGTG GCTCACGCCT GTAATCCCAG 481 CACTTTGGCC TCGTGC

TABLE XXIIA Nucleotide sequence alignment of 85P1B3 (SEQ ID NO: 702)with splice variant 1 (SEQ ID NO: 703). Score = 160 bits (83), Expect =3e − 36 Identities = 83/83 (100%) Strand = Plus/Plus 85P1B3: 524gctatctcttaaaaacaaaagccatagtaaatgcatcagagatggatattcaaaatgttc 583||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||gctatctcttaaaaacaaaagccatagtaaatgcatcagagatggatattcaaaatgttc 78 Vrnt 1:19 85P1B3: 584 ctctatcagaaaagattgcagag 606 ||||||||||||||||||||||| Vrnt1: 79 ctctatcagaaaagattgcagag 101

TABLE XXIIIA Amino acid sequence alignment of 85P1B3 (SEQ ID No; 704)and splice variant 1 (Portion of SEQ ID NO: 707). Score = 64.8 bits(135), Expect = 2e − 08 Identities = 28/29 (96%) Frame = +1/+3 85P1B3:526 YLLKTKAIVNASEMDIQNVPLSEKIAELK 612 YLLKTKAIVNASEMDIQNVPLSEKIAE+K Vrnt1: 21 YLLKTKAIVNASEMDIQNVPLSEKIAEVK 107

TABLE XXIVA Peptide sequences from the translation of the nucleotidesequence of splice variant 1. Open reading frame Amino acid sequencesFrame 1 FFFSYLAIS*KQKP**MHQRWIFKMFLYQKRLQR (SEQ ID NO: 705)*NFMMVVCFFKIQTTLDNFYQ*TWG**NGMMLNN PFYLI*PSLLNL**KTKY*NHTVRYSSGRKEKLLNCCTPAFSFVLGMDIIILP*RCLLCKYLVIAQLS QT*E**NSY*EFLAGRGGSRL*SQHFGLV Frame 2FFFPI*LSLKNKSHSKCIRDGYSKCSSIRKDCRG (SEQ ID NO: 706)KIS*WLYAFLKYRQLLITSTNELGDDEMA*CSII LFT*FDLPY*ICNEKQNTKTTL*GIVQEERKSCSTAALLHSPLCWEWISSSCHRGVFFANTL*LLNCL RHKSDETVIKNSWPGVVAHACNPSTLASC Frame 3FFFLSSYLLKTKAIVNASEMDIQNVPLSEKIAEV (SEQ ID NO: 707)KFHDGCMLF*NTDNS**LLPMNLGMMKWHDAQ*S FLLDLTFPIEFVMKNKILKPHCKV*FRKKGKAAQLLHSCILLCAGNGYHHLAIEVSSLQIPCNCSTVS DIRVMKQLLRIPGRAWWLTPVIPALWPRNote:Frame 3 gives the longest subsequence that is identical with 85P1B3amino acid sequence. In this Table each (*)indicates the product of asingle codon, i.e., a single unknown amino acid or a stop codon.

Example 39 Expression Analysis of 85P1B3 Splice Variants in NormalTissues and Patient Tumor Specimens

Expression of 85P1B3 described in Example 4 was performed using the85P1B3 SSH sequence as a probe. This nucleic acid sequence spans region701-1019 of the 85P1B3 gene, a region absent in the 85P1B3 splicevariant 1 (FIG. 19). Therefore, the Northern blots described in FIG. 11,FIG. 12, FIG. 13, FIG. 14, FIG. 15, FIG. 16, FIG. 17, and FIG. 18detected the transcript of 85P1B3 but not of splice variant 1.

A probe comprising region of homology between 85P1B3 and its splicevariant 1 is generated (Probe 1). This region spans nucleotide positions524-606 of 85P1B3 and 19-101 of splice variant 1. Normal tissue northernblots and patient cancer northern blots are probed with probe 1. Theresults have two bands, and show expression of the 1.2 kb transcript of85P1B3 and the transcript of its splice variant 1.

In another study, a probe comprising a region present in the ORF of thesplice variant but not in the ORF of 85P1B3 is generated (Probe 2). Thisregion spans nucleotide positions 102-496 of the splice variant 1.Normal tissue northern blots and patient cancer Northern blots areprobed with probe 2. The results have single bands, and show expressionof splice variant 1 but not the transcript of 85P1B3.

When 85P1B3 splice variant 1 is expressed in patient cancer specimens,and shows restricted expression in normal tissues, 85P1B3 splice variant1 is a suitable cancer target for cancer diagnosis and therapy.

Example 40 Splice Variant Protein Characteristics

The present variant protein is understood to be partial, and thus tocomprise domains of the full protein. Amino acids 7-35 of the 85P1B3variant 1 protein align with amino acids 172-200 of 85P1B3 with 96%identity, while the remaining downstream amino acids diverge from the85P1B3. This pattern of high homology to one section of the parentprotein coupled to a high divergence from the remaining portions of theparent protein form the hallmark of a splice variant.

Protein blast analysis of variant 1 shows that the 85P1B3 variant ishomologous to OIP5, a human protein known to be involved in adhesion andinvasion of epithelial cells (Brooks, G. F., et al., Mol Microbiol.(1991) 5:3063; Weel, J. F., et al., J Exp Med. (1991) 173:1395), with96% identity over 28 amino acids. Analysis by pFam or prosite failed toidentify any motifs. However motif homology was observed to Glyoxalase Iat aa 114-153 of the variant protein. Glyoxalase is aglutathione-mediating detoxifying enzyme, that protects cells fromadvanced glycation end products (AGEs) (Thornalley, P. J., Chem BiolInteract. (1998) 111:137). Glyoxalase is highly expressed in breastcancer cells (Rulli, A., et al., Breast Cancer Res Treat. (2001) 66:67).

Regarding localization, the 85P1B3 variant localizes to the cytoplasm(cytoplasmic 60.9% PSORT II) or the mitochondria (mitochondrial 0.519,PSORT).

Based on bioinformatic analysis (TMPred, Sosui) the 85P1B3 variant doesnot appear to contain transmembrane domains, but forms a solubleintracellular protein. Due to its homology to OIP5 and Glyoxalase I,85P1B3 is involved in the adhesion and invasion of epithelial cells, andhas a cancer-related expression pattern.

Example 41 Homology Comparison of 85P1B3 to Known Sequences

The 85P1B3 protein of FIG. 3 has 229 amino acids with calculatedmolecular weight of 24.69 kDa, and pI of 7.02. 85P1B3 is predicted to bea mitochondrial (60.9%) or cytoplasmic (21.7) protein.

85P1B3 shows best homology to human Opa interacting protein 5 (gi2815610) sharing 100% identity with that protein. Opacity associatedproteins (Opa) were identified in Neisseria gonorrhoeae as outermembrane proteins that are involved in mediating the adhesion ofNeisseria to mammalian cells and the invasion of human epithelial cells(Brooks, G. F., et al., Mol Microbiol. (1991) 5:3063; Weel, J. F., etal., J Exp Med. (1991) 173:1395). OPA proteins bind to membraneproteins, such as CD66 and carcinoembryonic antigen related cellularmolecule (CEACAM), on the surface of human epithelial and mononuclearcells, thereby facilitating entry of Neisseria into mammalian host cells(Muenzner, P., et al., J. Biol. Chem. (2001) 276:24331; Chen, T., etal., J. Exp. Med. (1997) 185:1557). In order to delineate the role ofOpa in adherence and invasion of human cells, Williams, et al., used atwo yeast hybrid system to identify Opa interacting proteins (Williams,J. M., et al., Mol. Microbiol. (1998) 27:171). Screening a human cDNAlibrary for Opa interacting partners, they identified Opa interactingprotein 5 or OIP5. OIP5 is an intracellular, cytoplasmic protein withhomology to thyroid hormone receptor interacting protein-6 (TRIP6)(Williams, J. M., et al., Ann N Y Acad Sci. (1996) 797:288). TRIP6 is anintracellular signaling molecule that relays information to the nucleusthereby regulating gene expression (Zhao, M., et al., Gene Expr. (1999)207; Wang, Y., et al., Gene. (1999) 234:403).

This information indicates that 85P1B3 can play a role in the adhesionand invasion of epithelial cells into adjacent tissues and basementmembranes, and regulate transcription by transmitting cell surfacesignals to the nucleus.

Accordingly, when 85P1B3 functions as a regulator of cell adhesion andinvasion, or as a modulator of transcription involved in activatinggenes associated with tumorigenesis or in blocking expression of genesthat repress tumorigenesis, 85P1B3 is used for therapeutic, diagnostic,prognostic and/or preventative purposes.

Example 42 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (Abe, K,et al., J Neurochem. (2001) 76:217-223). In particular, OPA has beenreported to associate with a phosphatase and surface receptors (Hauck,C., et al., Infect. Immun. (1999) 67:5490; Muenzner, P., et al., J.Biol. Chem. (2001) 276:24331). Using immunoprecipitation and Westernblotting techniques, proteins are identified that associate with 85P1B3and mediate signaling events. Several pathways known to play a role incancer biology can be regulated by 85P1B3, including phospholipidpathways such as PI3K, AKT, etc., adhesion and migration pathways,including FAK, Rho, Rac-1, etc., as well as mitogenic/survival cascadessuch as ERK, p38, etc. (Mirza, A., et al., Cell Growth Differ. (2000)11:279-292; Janulis, M., J. Biol. Chem. (1999) 274:801-813; Ivanov, V.N., et al., Oncogene (2000) 19:3003-3012; Machesky, L. M., et al., J.Cell Biol. (1997) 138:913-926).

Using, e.g., Western blotting techniques the ability of 85P1B3 toregulate these pathways is confirmed. Cells expressing or lacking 85P1B3are either left untreated or stimulated with cytokines, androgen andanti-integrin antibodies. Cell lysates are analyzed usinganti-phospho-specific antibodies (Cell Signaling, Santa CruzBiotechnology) in order to detect phosphorylation and regulation of ERK,p38, AKT, P13K, PLC and other signaling molecules. When 85P1B3 plays arole in the regulation of signaling pathways, whether individually orcommunally, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

To confirm that 85P1B3 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

p53-luc, p53; SAPK; growth/differentiation/apoptosis

CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 85P1B3 are mapped and used for theidentification and validation of therapeutic targets. When 85P1B3 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 43 Involvement in Tumor Progression

The 85P1B3 gene can contribute to the growth of cancer cells. The roleof 85P1B3 in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate, colon, bladder and kidneycell lines, as well as NIH-3T3 cells engineered to stably express85P1B3. Parental cells lacking 85P1B3 and cells expressing 85P1B3 areevaluated for cell growth using a well-documented proliferation assay(Fraser, S. P., et al., Prostate (2000) 44:61, Johnson, D. E., et al.,Anticancer Drugs (1996) 7:288).

To confirm the role of 85P1B3 in the transformation process, its effectin colony forming assays is investigated. Parental NIH-3T3 cells lacking85P1B3 are compared to NIH-3T3 cells expressing 85P1B3, using a softagar assay under stringent and more permissive conditions (Song, Z., etal., Cancer Res. (2000) 60:6730).

To confirm the role of 85P1B3 in invasion and metastasis of cancercells, a well-established assay is used, e.g., a Transwell® InsertSystem assay (Becton Dickinson) (Krueger, S., Cancer Res. (1999)59:6010-6014). Control cells, including prostate, colon, bladder andkidney cell lines lacking 85P1B3 are compared to cells expressing85P1B3. Cells are loaded with the fluorescent dye, calcein, and platedin the top well of the Transwell® insert coated with a basement membraneanalog. Invasion is determined by fluorescence of cells in the lowerchamber relative to the fluorescence of the entire cell population.

85P1B3 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 85P1B3 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek, Z. A., J.Cell Physiol. (1988) 136:247). In short, cells are grown under bothoptimal (full serum) and limiting (low serum) conditions are labeledwith BrdU and stained with anti-BrdU Ab and propidium iodide. Cells areanalyzed for entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 85P1B3, including normal and tumorprostate, colon and lung cells. Engineered and parental cells aretreated with various chemotherapeutic agents, such as etoposide,flutamide, etc, and protein synthesis inhibitors, such as cycloheximide.Cells are stained with annexin V-FITC and cell death is measured by FACSanalysis. The modulation of cell death by 85P1B3 can play a criticalrole in regulating tumor progression and tumor load.

When 85P1B3 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 44 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan, D., et al., J. Cell. (1996) 86:353; Folkman, J.,Endocrinology (1998) 139:441). Several assays have been developed tomeasure angiogenesis in vitro and in vivo, such as the tissue cultureassays endothelial cell tube formation and endothelial cellproliferation. Using these assays as well as in vitroneo-vascularization, the role of 85P1B3 in angiogenesis, enhancement orinhibition, is confirmed.

For example, endothelial cells engineered to express 85P1B3 areevaluated using tube formation and proliferation assays. The effect of85P1B3 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 85P1B3 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. When85P1B3 affects angiogenesis, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 45 Regulation of Transcription

The cytoplasmic localization of 85P1B3 and its similarity to TRIP5support the use in accordance with the present invention of 85P1B3 as amodulator of the transcriptional regulation of eukaryotic genes.Regulation of gene expression is confirmed, e.g., by studying geneexpression in cells expressing or lacking 85P1B3. For this purpose, twotypes of experiments are performed.

In the first set of experiments, RNA from parental and 85P1B3-expressingcells are extracted and hybridized to commercially available gene arrays(Clontech) (Smid-Koopman, E., et al., Br. J. Cancer (2000) 83:246).Resting cells as well as cells treated with FBS or androgen arecompared. Differentially expressed genes are identified in accordancewith procedures known in the art. The differentially expressed genes arethen mapped to biological pathways (Chen, K., et al., Thyroid (2001)11:41).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

When 85P1B3 plays a role in gene regulation, it is used as a target fordiagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46 Involvement in Cell Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. 85P1B3 can participate in cellular organization, and as aconsequence cell adhesion and motility. This is supported by thepresence of an RGD motif in the N-terminal portion of 85P1B3 (see TableXIX). To confirm that 85P1B3 regulates cell adhesion, control cellslacking 85P1B3 are compared to cells expressing 85P1B3, using techniquespreviously described (see, e.g., Haier, et al., Br. J. Cancer (1999)80:1867; Lehr, et al., J. Natl. Cancer Inst. (1998) 90:118). Briefly, inone embodiment, cells labeled with a fluorescent indicator, such ascalcein, are incubated on tissue culture wells coated with media aloneor with matrix proteins. Adherent cells are detected by fluorimetricanalysis and percent adhesion is calculated. In another embodiment,cells lacking or expressing 85P1B3 are analyzed for their ability tomediate cell-cell adhesion using similar experimental techniques asdescribed above. Both of these experimental systems are used to identifyproteins, antibodies and/or small molecules that modulate cell adhesionto extracellular matrix and cell-cell interaction. Since cell adhesionplays a critical role in tumor growth, progression, and, colonization,when 85P1B3 is involved in these processes it serves as a diagnostic,prognostic, preventative and/or therapeutic modality.

Example 47 Protein-Protein Association

Two proteins with homology to 85P1B3, namely OIP5 and TRIP6, have beenshown to interact with other proteins, thereby regulating signaltransduction, gene transcription, and cell adhesion. Usingimmunoprecipitation techniques as well as two yeast hybrid systems,proteins are identified that associate with 85P1B3. Immunoprecipitatesfrom cells expressing 85P1B3 and cells lacking 85P1B3 are compared forspecific protein-protein associations.

Studies are performed to confirm the extent of association of 85P1B witheffector molecules, such as receptors, adaptor proteins andSH2-containing proteins. Studies comparing 85P1B3 positive and 85P1B3negative cells as well as studies comparing unstimulated/resting cellsand cells treated with epithelial cell activators, such as cytokines,growth factors, androgen and anti-integrin Ab reveal uniqueinteractions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Drees, B. L., Curr. Opin. Chem. Biol. (1999)3:64-70). A vector carrying a library of proteins fused to theactivation domain of a transcription factor is introduced into yeastexpressing a 85P1B3-DNA-binding domain fusion protein and a reporterconstruct. Protein-protein interaction is detected by colorimetricreporter activity. Specific association with effector molecules andtranscription factors directs one of skill to the mode of action of85P1B3, and thus identifies therapeutic, prognostic, preventative and/ordiagnostic targets for cancer. This and similar assays are also used toidentify and screen for small molecules that interact with 85P1B3.

When 85P1B3 associates with proteins or small molecules it is used as atarget for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Throughout this application, various website data content, publications,patent applications and patents are referenced. The disclosures of eachof these references are hereby incorporated by reference herein in theirentireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables TABLE I Tissues that Express 85P1B3 When Malignant ProstateBladder Kidney Colon Lung Ovary Breast Stomach Uterus Cervix

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 0−2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1−3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −20 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0−3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2−3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3−3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4−3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4−3 −1 V 11 2 W 7 Y

TABLE IV A POSITION POSITION POSITION C Terminus 2 (Primary 3 (Primary(Primary Anchor) Anchor) Anchor) SUPERMOTIFS A1 TI LVMS FWY (SEQ ID NO:745) A2 LIVM ATQ IV MATL (SEQ ID NO: 746) (SEQ ID NO: 747) A3 VSMA TLIRK (SEQ ID NO: 748) A24 YF WIVLMT FI YWLM (SEQ ID NO: 749) (SEQ ID NO:750) B7 P VILF MWYA (SEQ ID NO: 751) B27 RHK FYL WMIVA (SEQ ID NO: 752)B44 E D FWYLIMVA (SEQ ID NO: 753) B58 ATS FWY LIVMA (SEQ ID NO: 754) B62QL IVMP FWYMIVLA (SEQ ID NO: 755) (SEQ ID NO: 756) MOTIFS A1 TSM Y A1 DEAS Y (SEQ ID NO: 868) A2.1 LM VQIAT V LIMAT (SEQ ID NO: 757) (SEQ ID NO:758) A3 LMVISATF CGD KYR HFA (SEQ ID NO: 759) (SEQ ID NO: 760) A11VTMLISAGN CDF K RYH (SEQ ID NO: 761) (SEQ ID NO: 762) A24 YF WM FLIW(SEQ ID NO: 763) (SEQ ID NO: 764) A*3101 MVT ALIS R K (SEQ ID NO: 765)A*3301 MVALF IST RK (SEQ ID NO: 766) A*6801 AVT MSLI RK (SEQ ID NO: 767)B*0702 P LMF WYAIV (SEQ ID NO: 768) B*3501 P LMFWY IVA (SEQ ID NO: 769)B51 P LIVF WYAM (SEQ ID NO: 770) B*5301 P IMFWY ALV (SEQ ID NO: 771)B*5401 P ATIV LMFWY (SEQ ID NO: 772)Bolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table.

Bolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table. TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I,L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferredFMYLIVW M T I VSTCPALIM MH MH (SEQ ID NO: 773) (SEQ ID NO: 774)deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM (SEQ IDNO: 775) (SEQ ID NO: 776) (SEQ ID NO: 777) deleterious C CH FD CWD GDE DDR7 preferred MFLIVWY M W A IVMSACTPL M IV (SEQ ID NO: 778) (SEQ ID NO:779) deleterious C G GRD N G DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1°anchor 6 motif a LIVMFY D preferred (SEQ ID NO: 780) motif b LIVMFAYDNQEST KRH preferred (SEQ ID NO: 781) (SEQ ID NO: 782) DR MFLIVWYVMSTACPLI Supermotif (SEQ ID NO: 783) (SEQ ID NO: 784)Italicized residues indicate less preferred or “tolerated” residues.

TABLE IV D POSITION SUPER MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor1° Anchor TILVMS FWY (SEQ ID NO: 785) A2 1° Anchor 1° Anchor LIVMATQLIVMAT (SEQ ID (SEQ ID NO: 786) NO: 787) A3 preferred 1° Anchor YFW YFWYFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK (SEQ ID NO: 788)deleterious DE (3/5); DE P (5/5) (4/5) A24 1° Anchor 1° Anchor YFWIVLMTFIYWLM (SEQ ID (SEQ ID NO: 789) NO: 790) B7 preferred FWY (5/5) 1°Anchor FWY FWY 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA (SEQ ID (SEQID NO: 791) NO: 792) deleterious DE (3/5); DE G QN DE P(5/5); (3/5)(4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1° Anchor 1°Anchor RHKFYLWMIVA (SEQ ID NO: 793) B44 1° Anchor 1° Anchor ED FWYLIMVA (SEQ IDNO: 794) B58 1° Anchor 1° Anchor ATS FWYLIVMA (SEQ ID NO: 795) B62 1°Anchor 1° Anchor QLIVMP FWYMIVLA (SEQ ID (SEQ ID NO: 796) NO: 797)

TABLE IV E 9 or 1 2 3 4 5 6 7 8 C-terminus C-terminus A1 preferred GFYW1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer (SEQ ID STM (SEQ ID Y NO:798) NO: 799) deleterious DE RHKLIVMP A G A (SEQ ID NO: 800) A1preferred GRHK ASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer (SEQID (SEQ ID DEAS (SEQ ID (SEQ ID (SEQ ID Y NO: 801) NO: 802) (SEQ ID NO:804) NO: 805) NO: 806) NO: 803) deleterious A RHKDEPYFW DE PQN RHK PG GP(SEQ ID NO: 807) A1 preferred YFW 1° Anchor DEAQN A YFWQN PASTC GDE P 1°Anchor 10-mer STM (SEQ ID (SEQ ID (SEQ ID Y NO: 808) NO: 809) NO: 810)deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A (SEQ ID (SEQ ID NO: 811)NO: 812) A1 preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anchor10-mer (SEQ ID DEAS Y NO: 813) (SEQ ID NO: 814) deleterious RHKRHKDEPYFW P G PRHK QN (SEQ ID (SEQ ID NO: 815) NO: 816) A2.1 preferredYFW 1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT VLIMAT (SEQ ID(SEQ ID NO: 817) NO: 818) deleterious DEP DERKH RKH DERKH (SEQ ID (SEQID NO: 819) NO: 820) A2.1 preferred AYFW 1° Anchor LVIM G G FYWLVIM 1°Anchor 10-mer LMIVQAT (SEQ ID (SEQ ID VLIMAT (SEQ ID NO: 822) NO: 823)(SEQ ID NO: 821) NO: 824) deleterious DEP DE RKHA P RKH DERKH RKH (SEQID (SEQ ID NO: 825) NO: 826) A3c preferred RHK 1° Anchor YFW PRHKYFW AYFW P 1° Anchor LMVISATFCGD (SEQ ID KYRHFA (SEQ ID NO: 828) (SEQ ID NO:827) NO: 829) deleterious DEP DE A11 preferred A 1° Anchor YFW YFW A YFWYFW P 1° Anchor VTLMISAGNCDF KRYH (SEQ ID (SEQ ID NO: 830) NO: 831)deleterious DEP A G A24 preferred YFWRHK 1° Anchor STC YFW YFW 1° Anchor9-mer (SEQ ID YFWM FLIW NO: 832) (SEQ ID (SEQ ID NO: 833) NO: 834)deleterious DEG DE G QNP DERHK G AQN (SEQ ID NO: 835) A24 preferred 1°Anchor P YFWP P 1° Anchor 10-mer YFWM (SEQ ID FLIW (SEQ ID NO: 837) (SEQID NO: 836) NO: 838) deleterious GDE QN RHK DE A QN DEA A3101 preferredRHK 1° Anchor YFW P YFW YFW AP 1° Anchor MVTALIS RK (SEQ ID NO: 839)deleterious DEP DE ADE DE DE DE A3301 preferred 1° Anchor YFW AYFW 1°Anchor MVALFIST (SEQ ID RK (SEQ ID NO: 841) NO: 840) deleterious GP DEA6801 preferred YFWSTC 1° Anchor YFWLIVM YFW P 1° Anchor (SEQ ID AVTMSLI(SEQ ID RK NO: 842) (SEQ ID NO: 844) NO: 843) deleterious GP DEG RHK AB0702 preferred RHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor (SEQ ID PLMFWYAIV NO: 845) (SEQ ID NO: 846) deleterious DEQNP DEP DE DE GDE QN DE(SEQ ID NO: 847) B3501 preferred FWYLIVM 1° Anchor FWY FWY 1° Anchor(SEQ ID P LMFWYIVA NO: 848) (SEQ ID NO: 849) deleterious AGP G G B51preferred LIVMFWY 1° Anchor FWY STC FWY G FWY 1° Anchor (SEQ ID PLIVFWYAM NO: 850) (SEQ ID NO: 851) deleterious AGPDER DE G DEQN GDEHKSTC (SEQ ID (SEQ ID NO: 853) NO: 852) B5301 preferred LIVMFWY 1°Anchor FWY STC FWY LIVMFWY FWY 1° Anchor (SEQ ID P (SEQ ID IMFWYALV NO:854) NO: 855) (SEQ ID NO: 856) deleterious AGPQN G RHKQN DE (SEQ ID (SEQID NO: 857) NO: 858) B5401 preferred FWY 1° Anchor FWYLIVM LIVM ALIVMFWYAP 1°Anchor P (SEQ ID (SEQ ID (SEQ ID (SEQ ID ATIVLMFWY NO: 859) NO:860) NO: 861) NO: 862) (SEQ ID NO: 863) deleterious GPQNDE GDESTC RHKDEDE QNDGE DE (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 864) NO: 865) NO: 866)NO: 867)Italicized residues indicate less preferred or “tolerated” residues. Theinformation in this Table is specific for 9-mers unless otherwisespecified.

TABLE V HLA Peptide Scoring Results - 85P1B3 - A1, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 114 VLEAPFLVG 4.500 1 2 192 LSEKIAELK 2.700 2 3 87 LADSVHLAW 2.5003 4 27 AIDQASFTT 2.500 4 5 164 LSSDKMVCY 1.500 5 6 217 LSEVTPDQS 1.350 67 182 ASEMDIQNV 1.350 7 8 12 CATPPRGDF 1.000 8 9 122 GIEGSLKGS 0.900 910 196 IAELKEKIV 0.900 10 11 141 GIPVGFHLY 0.500 11 12 100 SLGAVVFSR0.500 12 13 184 EMDIQNVPL 0.500 13 14 57 AEEPAAGPQ 0.450 14 15 36SMEWDTQVV 0.450 15 16 46 GSSPLGPAG 0.300 16 17 138 GSCGIPVGF 0.300 17 1813 ATPPRGDFC 0.250 18 19 221 TPDQSKPEN 0.250 19 20 23 GTERAIDQA 0.225 2021 61 AAGPQLPSW 0.200 21 22 120 LVGIEGSLK 0.200 22 23 169 MVCYLLKTK0.200 23 24 203 IVLTHNRLK 0.200 24 25 56 GAEEPAAGP 0.180 25 26 130STYNLLFCG 0.125 26 27 128 KGSTYNLLF 0.125 27 28 140 CGIPVGFHL 0.125 2829 124 EGSLKGSTY 0.125 29 30 109 VTNNVVLEA 0.125 30 31 1 MAAQPLRHR 0.10031 32 2 AAQPLRHRS 0.100 32 33 69 WLQPERCAV 0.100 33 34 154 ALAALRGHF0.100 34 35 165 SSDKMVCYL 0.075 35 36 31 ASFTTSMEW 0.075 36 37 129GSTYNLLFC 0.075 37 38 149 YSTHAALAA 0.075 38 39 66 LPSWLQPER 0.050 39 40136 FCGSCGIPV 0.050 40 41 111 NNVVLEAPF 0.050 41 42 150 STHAALAAL 0.05042 43 167 DKMVCYLLK 0.050 43 44 49 PLGPAGLGA 0.050 44 45 204 VLTHNRLKS0.050 45 46 163 CLSSDKMVC 0.050 46 47 38 EWDTQVVKG 0.050 47 48 152HAALAALRG 0.050 48 49 179 IVNASEMDI 0.050 49 50 181 NASEMDIQN 0.050 50

TABLE VI HLA Peptide Scoring Results - 85P1B3 - A1, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 217 LSEVTPDQSK 27.000 51 2 36 SMEWDTQVVK 18.000 52 3 196IAELKEKIVL 4.500 53 4 69 WLQPERCAVF 2.000 54 5 114 VLEAPFLVGI 1.800 55 617 RGDFCGGTER 1.250 56 7 140 CGIPVGFHLY 1.250 57 8 13 ATPPRGDFCG 1.25058 9 163 CLSSDKMVCY 1.000 59 10 2 AAQPLRHRSR 1.000 60 11 56 GAEEPAAGPQ0.900 61 12 57 AEEPAAGPQL 0.900 62 13 122 GIEGSLKGST 0.900 63 14 99RSLGAVVFSR 0.750 64 15 27 AIDQASFTTS 0.500 65 16 90 SVHLAWDLSR 0.500 6617 184 EMDIQNVPLS 0.500 67 18 150 STHAALAALR 0.500 68 19 46 GSSPLGPAGL0.300 69 20 23 GTERAIDQAS 0.225 70 21 119 FLVGIEGSLK 0.200 71 22 202KIVLTHNRLK 0.200 72 23 186 DIQNVPLSEK 0.200 73 24 65 QLPSWLQPER 0.200 7425 165 SSDKMVCYLL 0.150 75 26 182 ASEMDIQNVP 0.135 76 27 94 AWDLSRSLGA0.125 77 28 71 QPERCAVFQC 0.113 78 29 87 LADSVHLAWD 0.100 79 30 12CATPPRGDFC 0.100 80 31 11 RCATPPRGDF 0.100 81 32 153 AALAALRGHF 0.100 8233 61 AAGPQLPSWL 0.100 83 34 168 KMVCYLLKTK 0.100 84 35 129 GSTYNLLFCG0.075 85 36 192 LSEKIAELKE 0.068 86 37 116 EAPFLVGIEG 0.050 87 38 155LAALRGHFCL 0.050 88 39 203 IVLTHNRLKS 0.050 89 40 112 NVVLEAPFLV 0.05090 41 139 SCGIPVGFHL 0.050 91 42 178 AIVNASEMDI 0.050 92 43 26RAIDQASFTT 0.050 93 44 159 RGHFCLSSDK 0.050 94 45 110 TNNVVLEAPF 0.05095 46 108 RVTNNVVLEA 0.050 96 47 30 QASFTTSMEW 0.050 97 48 113VVLEAPFLVG 0.050 98 49 120 LVGIEGSLKG 0.050 99 50 137 CGSCGIPVGF 0.050100

TABLE VII HLA Peptide Scoring Results - 85P1B3 - A2, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 113 VVLEAPFLV 910.291 101 2 212 SLMKILSEV 591.888 102 3 172YLLKTKAIV 485.348 103 4 69 WLQPERCAV 319.939 104 5 86 VLADSVHLA 79.642105 6 134 LLFCGSCGI 65.622 106 7 168 KMVCYLLKT 43.325 107 8 78 FQCAQCHAV32.438 108 9 119 FLVGIEGSL 12.775 109 10 112 NVVLEAPFL 10.281 110 11 202KIVLTHNRL 10.281 111 12 195 KIAELKEKI 10.087 112 13 162 FCLSSDKMV 7.727113 14 85 AVLADSVHL 6.916 114 15 35 TSMEWDTQV 6.887 115 16 156 AALRGHFCL6.367 116 17 54 GLGAEEPAA 4.968 117 18 191 PLSEKIAEL 4.432 118 19 33FTTSMEWDT 3.571 119 20 93 LAWDLSRSL 3.433 120 21 115 LEAPFLVGI 3.014 12122 27 AIDQASFTT 2.377 122 23 26 RAIDQASFT 2.334 123 24 147 HLYSTHAAL2.324 124 25 136 FCGSCGIPV 2.088 125 26 163 CLSSDKMVC 2.037 126 27 42QVVKGSSPL 1.869 127 28 96 DLSRSLGAV 1.560 128 29 179 IVNASEMDI 1.552 12930 101 LGAVVFSRV 1.466 130 31 36 SMEWDTQVV 1.318 131 32 205 LTHNRLKSL1.160 132 33 140 CGIPVGFHL 0.809 133 34 62 AGPQLPSWL 0.767 134 35 126SLKGSTYNL 0.748 135 36 165 SSDKMVCYL 0.706 136 37 209 RLKSLMKIL 0.705137 38 150 STHAALAAL 0.682 138 39 155 LAALRGHFC 0.645 139 40 197AELKEKIVL 0.630 140 41 184 EMDIQNVPL 0.463 141 42 129 GSTYNLLFC 0.410142 43 133 NLLFCGSCG 0.276 143 44 132 YNLLFCGSC 0.273 144 45 109VTNNVVLEA 0.270 145 46 177 KAIVNASEM 0.242 146 47 100 SLGAVVFSR 0.199147 48 13 ATPPRGDFC 0.186 148 49 198 ELKEKIVLT 0.184 149 50 189NVPLSEKIA 0.178 150

TABLE VIII HLA Peptide Scoring Results - 85P1B3 - A2, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 100 SLGAVVFSRV 132.149 151 2 204 VLTHNRLKSL 83.527 152 3 195KIAELKEKIV 56.266 153 4 133 NLLFCGSCGI 38.601 154 5 104 VVFSRVTNNV38.280 155 6 112 NVVLEAPFLV 35.298 156 7 154 ALAALRGHFC 27.324 157 8 211KSLMKILSEV 13.523 158 9 164 LSSDKMVCYL 12.295 159 10 114 VLEAPFLVGI9.921 160 11 181 NASEMDIQNV 9.109 161 12 183 SEMDIQNVPL 6.301 162 13 212SLMKILSEVT 5.539 163 14 78 FQCAQCHAVL 4.085 164 15 85 AVLADSVHLA 3.699165 16 155 LAALRGHFCL 2.925 166 17 35 TSMEWDTQVV 2.824 167 18 26RAIDQASFTT 2.461 168 19 41 TQVVKGSSPL 2.166 169 20 96 DLSRSLGAVV 2.139170 21 190 VPLSEKIAEL 2.017 171 22 61 AAGPQLPSWL 1.632 172 23 76AVFQCAQCHA 1.608 173 24 149 YSTHAALAAL 1.475 174 25 128 KGSTYNLLFC 1.436175 26 178 AIVNASEMDI 1.435 176 27 197 AELKEKIVLT 1.233 177 28 126SLKGSTYNLL 1.122 178 29 108 RVTNNVVLEA 1.000 179 30 34 TTSMEWDTQV 0.966180 31 187 IQNVPLSEKI 0.881 181 32 139 SCGIPVGFHL 0.809 182 33 111NNVVLEAPFL 0.767 183 34 169 MVCYLLKTKA 0.739 184 35 173 LLKTKAIVNA 0.680185 36 147 HLYSTHAALA 0.541 186 37 172 YLLKTKAIVN 0.520 187 38 86VLADSVHLAW 0.519 188 39 125 GSLKGSTYNL 0.516 189 40 3 AQPLRHRSRC 0.504190 41 95 WDLSRSLGAV 0.492 191 42 170 VCYLLKTKAI 0.370 192 43 216ILSEVTPDQS 0.255 193 44 146 FHLYSTHAAL 0.252 194 45 162 FCLSSDKMVC 0.226195 46 88 ADSVHLAWDL 0.223 196 47 82 QCHAVLADSV 0.222 197 48 141GIPVGFHLYS 0.214 198 49 144 VGFHLYSTHA 0.204 199 50 37 MEWDTQVVKG 0.193200

TABLE IX HLA Peptide Scoring Results - 85P1B3 - A3, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 100 SLGAVVFSR 54.000 201 2 168 KMVCYLLKT 4.050 202 3 141 GIPVGFHLY3.600 203 4 147 HLYSTHAAL 3.000 204 5 134 LLFCGSCGI 3.000 205 6 126SLKGSTYNL 2.700 206 7 120 LVGIEGSLK 2.000 207 8 169 MVCYLLKTK 1.500 2089 187 IQNVPLSEK 1.350 209 10 212 SLMKILSEV 0.675 210 11 119 FLVGIEGSL0.608 211 12 54 GLGAEEPAA 0.600 212 13 154 ALAALRGHF 0.600 213 14 86VLADSVHLA 0.600 214 15 209 RLKSLMKIL 0.450 215 16 163 CLSSDKMVC 0.400216 17 160 GHFCLSSDK 0.300 217 18 69 WLQPERCAV 0.300 218 19 172YLLKTKAIV 0.300 219 20 37 MEWDTQVVK 0.300 220 21 203 IVLTHNRLK 0.300 22122 202 KIVLTHNRL 0.270 222 23 195 KIAELKEKI 0.270 223 24 36 SMEWDTQVV0.200 224 25 184 EMDIQNVPL 0.180 225 26 157 ALRGHFCLS 0.180 226 27 114VLEAPFLVG 0.180 227 28 192 LSEKIAELK 0.150 228 29 191 PLSEKIAEL 0.135229 30 113 VVLEAPFLV 0.135 230 31 218 SEVTPDQSK 0.135 231 32 179IVNASEMDI 0.120 232 33 76 AVFQCAQCH 0.100 233 34 213 LMKILSEVT 0.100 23435 70 LQPERCAVF 0.090 235 36 65 QLPSWLQPE 0.090 236 37 85 AVLADSVHL0.090 237 38 42 QVVKGSSPL 0.090 238 39 112 NVVLEAPFL 0.090 239 40 109VTNNVVLEA 0.090 240 41 207 HNRLKSLMK 0.080 241 42 204 VLTHNRLKS 0.080242 43 138 GSCGIPVGF 0.068 243 44 198 ELKEKIVLT 0.068 244 45 18GDFCGGTER 0.060 245 46 92 HLAWDLSRS 0.060 246 47 49 PLGPAGLGA 0.060 24748 216 ILSEVTPDQ 0.045 248 49 23 GTERAIDQA 0.045 249 50 150 STHAALAAL0.045 250

TABLE X HLA Peptide Scoring Results - 85P1B3 - A3, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 168 KMVCYLLKTK 67.500 251 2 119 FLVGIEGSLK 45.000 252 3 36SMEWDTQVVK 20.000 253 4 163 CLSSDKMVCY 6.000 254 5 191 PLSEKIAELK 4.500255 6 65 QLPSWLQPER 4.000 256 7 69 WLQPERCAVF 3.000 257 8 114 VLEAPFLVGI2.700 258 9 90 SVHLAWDLSR 2.400 259 10 186 DIQNVPLSEK 1.350 260 11 147HLYSTHAALA 1.000 261 12 100 SLGAVVFSRV 0.900 262 13 202 KIVLTHNRLK 0.900263 14 126 SLKGSTYNLL 0.900 264 15 133 NLLFCGSCGI 0.900 265 16 198ELKEKIVLTH 0.810 266 17 99 RSLGAVVFSR 0.608 267 18 86 VLADSVHLAW 0.600268 19 204 VLTHNRLKSL 0.450 269 20 157 ALRGHFCLSS 0.360 270 21 173LLKTKAIVNA 0.300 271 22 154 ALAALRGHFC 0.200 272 23 150 STHAALAALR 0.200273 24 108 RVTNNVVLEA 0.180 274 25 178 AIVNASEMDI 0.180 275 26 104VVFSRVTNNV 0.150 276 27 212 SLMKILSEVT 0.150 277 28 217 LSEVTPDQSK 0.150278 29 76 AVFQCAQCHA 0.100 279 30 85 AVLADSVHLA 0.090 280 31 112NVVLEAPFLV 0.090 281 32 209 RLKSLMKILS 0.080 282 33 141 GIPVGFHLYS 0.072283 34 172 YLLKTKAIVN 0.060 284 35 96 DLSRSLGAVV 0.060 285 36 54GLGAEEPAAG 0.060 286 37 92 HLAWDLSRSL 0.060 287 38 216 ILSEVTPDQS 0.060288 39 195 KIAELKEKIV 0.045 289 40 193 SEKIAELKEK 0.045 290 41 113VVLEAPFLVG 0.041 291 42 125 GSLKGSTYNL 0.041 292 43 166 SDKMVCYLLK 0.040293 44 206 THNRLKSLMK 0.040 294 45 184 EMDIQNVPLS 0.036 295 46 200KEKIVLTHNR 0.036 296 47 134 LLFCGSCGIP 0.030 297 48 34 TTSMEWDTQV 0.030298 49 130 STYNLLFCGS 0.030 299 50 41 TQVVKGSSPL 0.027 300

TABLE XI HLA Peptide Scoring Results - 85P1B3 - A11, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 120 LVGIEGSLK 2.000 301 2 169 MVCYLLKTK 1.000 302 3 187 IQNVPLSEK0.600 303 4 203 IVLTHNRLK 0.300 304 5 100 SLGAVVFSR 0.240 305 6 160GHFCLSSDK 0.120 306 7 37 MEWDTQVVK 0.120 307 8 113 VVLEAPFLV 0.090 308 9218 SEVTPDQSK 0.090 309 10 207 HNRLKSLMK 0.080 310 11 76 AVFQCAQCH 0.040311 12 66 LPSWLQPER 0.040 312 13 179 IVNASEMDI 0.040 313 14 85 AVLADSVHL0.030 314 15 112 NVVLEAPFL 0.030 315 16 42 QVVKGSSPL 0.030 316 17 23GTERAIDQA 0.030 317 18 18 GDFCGGTER 0.024 318 19 167 DKMVCYLLK 0.024 31920 109 VTNNVVLEA 0.020 320 21 192 LSEKIAELK 0.020 321 22 202 KIVLTHNRL0.018 322 23 9 RSRCATPPR 0.012 323 24 91 VHLAWDLSR 0.012 324 25 3AQPLRHRSR 0.012 325 26 54 GLGAEEPAA 0.012 326 27 141 GIPVGFHLY 0.012 32728 195 KIAELKEKI 0.012 328 29 189 NVPLSEKIA 0.010 329 30 150 STHAALAAL0.010 330 31 156 AALRGHFCL 0.009 331 32 177 KAIVNASEM 0.009 332 33 212SLMKILSEV 0.008 333 34 126 SLKGSTYNL 0.008 334 35 147 HLYSTHAAL 0.008335 36 134 LLFCGSCGI 0.008 336 37 172 YLLKTKAIV 0.006 337 38 145GFHLYSTHA 0.006 338 39 70 LQPERCAVF 0.006 339 40 130 STYNLLFCG 0.006 34041 108 RVTNNVVLE 0.006 341 42 78 FQCAQCHAV 0.006 342 43 209 RLKSLMKIL0.006 343 44 119 FLVGIEGSL 0.006 344 45 205 LTHNRLKSL 0.005 345 46 194EKIAELKEK 0.005 346 47 87 LADSVHLAW 0.004 347 48 1 MAAQPLRHR 0.004 34849 154 ALAALRGHF 0.004 349 50 151 THAALAALR 0.004 350

TABLE XII HLA Peptide Scoring Results - 85P1B3 - A11, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 168 KMVCYLLKTK 0.900 351 2 90 SVHLAWDLSR 0.800 352 3 119FLVGIEGSLK 0.600 353 4 36 SMEWDTQVVK 0.400 354 5 150 STHAALAALR 0.200355 6 202 KIVLTHNRLK 0.180 356 7 186 DIQNVPLSEK 0.120 357 8 108RVTNNVVLEA 0.120 358 9 112 NVVLEAPFLV 0.090 359 10 65 QLPSWLQPER 0.080360 11 159 RGHFCLSSDK 0.060 361 12 99 RSLGAVVFSR 0.054 362 13 166SDKMVCYLLK 0.040 363 14 76 AVFQCAQCHA 0.040 364 15 191 PLSEKIAELK 0.040365 16 104 VVFSRVTNNV 0.040 366 17 206 THNRLKSLMK 0.040 367 18 200KEKIVLTHNR 0.036 368 19 85 AVLADSVHLA 0.030 369 20 193 SEKIAELKEK 0.030370 21 217 LSEVTPDQSK 0.020 371 22 169 MVCYLLKTKA 0.020 372 23 17RGDFCGGTER 0.012 373 24 178 AIVNASEMDI 0.012 374 25 205 LTHNRLKSLM 0.010375 26 34 TTSMEWDTQV 0.010 376 27 41 TQVVKGSSPL 0.009 377 28 86VLADSVHLAW 0.008 378 29 147 HLYSTHAALA 0.008 379 30 148 LYSTHAALAA 0.008380 31 133 NLLFCGSCGI 0.006 381 32 203 IVLTHNRLKS 0.006 382 33 78FQCAQCHAVL 0.006 383 34 187 IQNVPLSEKI 0.006 384 35 145 GFHLYSTHAA 0.006385 36 48 SPLGPAGLGA 0.006 386 37 155 LAALRGHFCL 0.006 387 38 11RCATPPRGDF 0.006 388 39 113 VVLEAPFLVG 0.006 389 40 139 SCGIPVGFHL 0.006390 41 195 KIAELKEKIV 0.006 391 42 171 CYLLKTKAIV 0.006 392 43 43VVKGSSPLGP 0.004 393 44 163 CLSSDKMVCY 0.004 394 45 100 SLGAVVFSRV 0.004395 46 135 LFCGSCGIPV 0.004 396 47 196 IAELKEKIVL 0.004 397 48 8HRSRCATPPR 0.004 398 49 189 NVPLSEKIAE 0.004 399 50 126 SLKGSTYNLL 0.004400

TABLE XIII HLA Peptide Scoring Results - 85P1B3 - A24, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 171 CYLLKTKAI 75.000 401 2 202 KIVLTHNRL 14.400 402 3 131TYNLLFCGS 10.800 403 4 140 CGIPVGFHL 10.080 404 5 209 RLKSLMKIL 9.600405 6 119 FLVGIEGSL 8.400 406 7 62 AGPQLPSWL 7.200 407 8 89 DSVHLAWDL7.200 408 9 112 NVVLEAPFL 6.000 409 10 42 QVVKGSSPL 6.000 410 11 47SSPLGPAGL 6.000 411 12 85 AVLADSVHL 6.000 412 13 156 AALRGHFCL 6.000 41314 93 LAWDLSRSL 5.760 414 15 148 LYSTHAALA 5.000 415 16 126 SLKGSTYNL4.000 416 17 184 EMDIQNVPL 4.000 417 18 147 HLYSTHAAL 4.000 418 19 79QCAQCHAVL 4.000 419 20 165 SSDKMVCYL 4.000 420 21 205 LTHNRLKSL 4.000421 22 150 STHAALAAL 4.000 422 23 128 KGSTYNLLF 4.000 423 24 70LQPERCAVF 3.600 424 25 111 NNVVLEAPF 3.600 425 26 195 KIAELKEKI 3.168426 27 138 GSCGIPVGF 2.800 427 28 161 HFCLSSDKM 2.750 428 29 12CATPPRGDF 2.400 429 30 154 ALAALRGHF 2.400 430 31 188 QNVPLSEKI 2.376431 32 177 KAIVNASEM 1.650 432 33 179 IVNASEMDI 1.500 433 34 134LLFCGSCGI 1.000 434 35 20 FCGGTERAI 1.000 435 36 105 VFSRVTNNV 0.840 43637 77 VFQCAQCHA 0.750 437 38 197 AELKEKIVL 0.600 438 39 107 SRVTNNVVL0.600 439 40 58 EEPAAGPQL 0.600 440 41 166 SDKMVCYLL 0.560 441 42 191PLSEKIAEL 0.528 442 43 29 DQASFTTSM 0.500 443 44 145 GFHLYSTHA 0.500 44445 19 DFCGGTERA 0.500 445 46 127 LKGSTYNLL 0.480 446 47 26 RAIDQASFT0.360 447 48 175 KTKAIVNAS 0.336 448 49 168 KMVCYLLKT 0.330 449 50 99RSLGAVVFS 0.300 450

TABLE XIV HLA Peptide Scoring Results - 85P1B3 - A24, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 171 CYLLKTKAIV 7.500 451 2 131 TYNLLFCGSC 7.500 452 3 190VPLSEKIAEL 6.600 453 4 196 IAELKEKIVL 6.000 454 5 41 TQVVKGSSPL 6.000455 6 125 GSLKGSTYNL 6.000 456 7 111 NNVVLEAPFL 6.000 457 8 84HAVLADSVHL 6.000 458 9 61 AAGPQLPSWL 5.760 459 10 139 SCGIPVGFHL 5.600460 11 165 SSDKMVCYLL 5.600 461 12 148 LYSTHAALAA 5.000 462 13 19DFCGGTERAI 5.000 463 14 46 GSSPLGPAGL 4.800 464 15 92 HLAWDLSRSL 4.800465 16 126 SLKGSTYNLL 4.800 466 17 164 LSSDKMVCYL 4.800 467 18 118PFLVGIEGSL 4.200 468 19 155 LAALRGHFCL 4.000 469 20 11 RCATPPRGDF 4.000470 21 78 FQCAQCHAVL 4.000 471 22 149 YSTHAALAAL 4.000 472 23 106FSRVTNNVVL 4.000 473 24 204 VLTHNRLKSL 4.000 474 25 110 TNNVVLEAPF 3.600475 26 69 WLQPERCAVF 3.600 476 27 153 AALAALRGHF 3.600 477 28 137CGSCGIPVGF 2.800 478 29 97 LSRSLGAVVF 2.000 479 30 187 IQNVPLSEKI 1.980480 31 178 AIVNASEMDI 1.500 481 32 114 VLEAPFLVGI 1.500 482 33 133NLLFCGSCGI 1.500 483 34 207 HNRLKSLMKI 1.100 484 35 170 VCYLLKTKAI 1.000485 36 77 VFQCAQCHAV 0.750 486 37 183 SEMDIQNVPL 0.720 487 38 208NRLKSLMKIL 0.720 488 39 201 EKIVLTHNRL 0.720 489 40 57 AEEPAAGPQL 0.720490 41 146 FHLYSTHAAL 0.600 491 42 105 VFSRVTNNVV 0.600 492 43 205LTHNRLKSLM 0.600 493 44 135 LFCGSCGIPV 0.500 494 45 161 HFCLSSDKMV 0.500495 46 145 GFHLYSTHAA 0.500 496 47 32 SFTTSMEWDT 0.500 497 48 88ADSVHLAWDL 0.480 498 49 211 KSLMKILSEV 0.462 499 50 26 RAIDQASFTT 0.360500

TABLE XV HLA Peptide Scoring Results - 85P1B3 - B7, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 85 AVLADSVHL 60.000 501 2 156 AALRGHFCL 36.000 502 3 112 NVVLEAPFL20.000 503 4 42 QVVKGSSPL 20.000 504 5 62 AGPQLPSWL 12.000 505 6 93LAWDLSRSL 12.000 506 7 150 STHAALAAL 4.000 507 8 89 DSVHLAWDL 4.000 5089 202 KIVLTHNRL 4.000 509 10 147 HLYSTHAAL 4.000 510 11 126 SLKGSTYNL4.000 511 12 79 QCAQCHAVL 4.000 512 13 205 LTHNRLKSL 4.000 513 14 140CGIPVGFHL 4.000 514 15 119 FLVGIEGSL 4.000 515 16 47 SSPLGPAGL 4.000 51617 209 RLKSLMKIL 4.000 517 18 177 KAIVNASEM 3.000 518 19 4 QPLRHRSRC3.000 519 20 179 IVNASEMDI 2.000 520 21 97 LSRSLGAVV 2.000 521 22 106FSRVTNNVV 2.000 522 23 184 EMDIQNVPL 1.200 523 24 165 SSDKMVCYL 1.200524 25 197 AELKEKIVL 1.200 525 26 113 VVLEAPFLV 1.000 526 27 29DQASFTTSM 1.000 527 28 35 TSMEWDTQV 0.600 528 29 117 APFLVGIEG 0.600 52930 212 SLMKILSEV 0.600 530 31 157 ALRGHFCLS 0.600 531 32 189 NVPLSEKIA0.500 532 33 103 AVVFSRVTN 0.450 533 34 20 FCGGTERAI 0.400 534 35 134LLFCGSCGI 0.400 535 36 107 SRVTNNVVL 0.400 536 37 188 QNVPLSEKI 0.400537 38 127 LKGSTYNLL 0.400 538 39 191 PLSEKIAEL 0.400 539 40 166SDKMVCYLL 0.400 540 41 58 EEPAAGPQL 0.400 541 42 142 IPVGFHLYS 0.400 54243 195 KIAELKEKI 0.400 543 44 75 CAVFQCAQC 0.300 544 45 155 LAALRGHFC0.300 545 46 48 SPLGPAGLG 0.300 546 47 53 AGLGAEEPA 0.300 547 48 69WLQPERCAV 0.300 548 49 80 CAQCHAVLA 0.300 549 50 26 RAIDQASFT 0.300 550

TABLE XVI HLA Peptide Scoring Results - 85P1B3 - B7, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 190 VPLSEKIAEL 80.000 551 2 106 FSRVTNNVVL 40.000 552 3 61AAGPQLPSWL 36.000 553 4 84 HAVLADSVHL 12.000 554 5 155 LAALRGHFCL 12.000555 6 46 GSSPLGPAGL 4.000 556 7 111 NNVVLEAPFL 4.000 557 8 92 HLAWDLSRSL4.000 558 9 78 FQCAQCHAVL 4.000 559 10 204 VLTHNRLKSL 4.000 560 11 139SCGIPVGFHL 4.000 561 12 149 YSTHAALAAL 4.000 562 13 126 SLKGSTYNLL 4.000563 14 164 LSSDKMVCYL 4.000 564 15 41 TQVVKGSSPL 4.000 565 16 207HNRLKSLMKI 4.000 566 17 125 GSLKGSTYNL 4.000 567 18 196 IAELKEKIVL 3.600568 19 15 PPRGDFCGGT 2.000 569 20 48 SPLGPAGLGA 2.000 570 21 4QPLRHRSRCA 2.000 571 22 142 IPVGFHLYST 2.000 572 23 66 LPSWLQPERC 2.000573 24 85 AVLADSVHLA 1.500 574 25 76 AVFQCAQCHA 1.500 575 26 165SSDKMVCYLL 1.200 576 27 117 APFLVGIEGS 1.200 577 28 88 ADSVHLAWDL 1.200578 29 183 SEMDIQNVPL 1.200 579 30 178 AIVNASEMDI 1.200 580 31 112NVVLEAPFLV 1.000 581 32 205 LTHNRLKSLM 1.000 582 33 104 VVFSRVTNNV 1.000583 34 71 QPERCAVFQC 0.600 584 35 157 ALRGHFCLSS 0.600 585 36 181NASEMDIQNV 0.600 586 37 35 TSMEWDTQVV 0.600 587 38 59 EPAAGPQLPS 0.600588 39 169 MVCYLLKTKA 0.500 589 40 108 RVTNNVVLEA 0.500 590 41 3AQPLRHRSRC 0.450 591 42 201 EKIVLTHNRL 0.400 592 43 170 VCYLLKTKAI 0.400593 44 146 FHLYSTHAAL 0.400 594 45 133 NLLFCGSCGI 0.400 595 46 187IQNVPLSEKI 0.400 596 47 208 NRLKSLMKIL 0.400 597 48 57 AEEPAAGPQL 0.360598 49 53 AGLGAEEPAA 0.300 599 50 154 ALAALRGHFC 0.300 600

TABLE XVII HLA Peptide Scoring Results - 85P1B3 - B35, 9-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 164 LSSDKMVCY 20.000 601 2 177 KAIVNASEM 12.000 602 3 209RLKSLMKIL 6.000 603 4 93 LAWDLSRSL 6.000 604 5 89 DSVHLAWDL 5.000 605 6138 GSCGIPVGF 5.000 606 7 47 SSPLGPAGL 5.000 607 8 126 SLKGSTYNL 3.000608 9 12 CATPPRGDF 3.000 609 10 156 AALRGHFCL 3.000 610 11 106 FSRVTNNVV3.000 611 12 97 LSRSLGAVV 3.000 612 13 31 ASFTTSMEW 2.500 613 14 141GIPVGFHLY 2.000 614 15 128 KGSTYNLLF 2.000 615 16 142 IPVGFHLYS 2.000616 17 35 TSMEWDTQV 2.000 617 18 4 QPLRHRSRC 2.000 618 19 70 LQPERCAVF2.000 619 20 202 KIVLTHNRL 2.000 620 21 29 DQASFTTSM 2.000 621 22 124EGSLKGSTY 2.000 622 23 195 KIAELKEKI 1.600 623 24 165 SSDKMVCYL 1.500624 25 85 AVLADSVHL 1.500 625 26 112 NVVLEAPFL 1.500 626 27 61 AAGPQLPSW1.500 627 28 26 RAIDQASFT 1.200 628 29 62 AGPQLPSWL 1.000 629 30 119FLVGIEGSL 1.000 630 31 140 CGIPVGFHL 1.000 631 32 154 ALAALRGHF 1.000632 33 42 QVVKGSSPL 1.000 633 34 150 STHAALAAL 1.000 634 35 205LTHNRLKSL 1.000 635 36 99 RSLGAVVFS 1.000 636 37 79 QCAQCHAVL 1.000 63738 147 HLYSTHAAL 1.000 638 39 111 NNVVLEAPF 1.000 639 40 221 TPDQSKPEN0.600 640 41 198 ELKEKIVLT 0.600 641 42 181 NASEMDIQN 0.600 642 43 175KTKAIVNAS 0.600 643 44 125 GSLKGSTYN 0.500 644 45 129 GSTYNLLFC 0.500645 46 149 YSTHAALAA 0.500 646 47 182 ASEMDIQNV 0.450 647 48 87LADSVHLAW 0.450 648 49 113 VVLEAPFLV 0.400 649 50 20 FCGGTERAI 0.400 650

TABLE XVIII HLA Peptide Scoring Results - 85P1B3 - B35, 10-mers Score(Estimate of Half Time of Disasso- Subsequence ciation of a MoleculeStart Residue Containing This Seq. Rank Position Listing Subsequence)ID# 1 190 VPLSEKIAEL 20.000 651 2 106 FSRVTNNVVL 15.000 652 3 97LSRSLGAVVF 15.000 653 4 164 LSSDKMVCYL 10.000 654 5 46 GSSPLGPAGL 5.000655 6 149 YSTHAALAAL 5.000 656 7 125 GSLKGSTYNL 5.000 657 8 84HAVLADSVHL 4.500 658 9 153 AALAALRGHF 3.000 659 10 61 AAGPQLPSWL 3.000660 11 35 TSMEWDTQVV 3.000 661 12 155 LAALRGHFCL 3.000 662 13 126SLKGSTYNLL 3.000 663 14 117 APFLVGIEGS 2.000 664 15 140 CGIPVGFHLY 2.000665 16 59 EPAAGPQLPS 2.000 666 17 205 LTHNRLKSLM 2.000 667 18 4QPLRHRSRCA 2.000 668 19 66 LPSWLQPERC 2.000 669 20 142 IPVGFHLYST 2.000670 21 163 CLSSDKMVCY 2.000 671 22 211 KSLMKILSEV 2.000 672 23 11RCATPPRGDF 2.000 673 24 48 SPLGPAGLGA 2.000 674 25 181 NASEMDIQNV 1.800675 26 30 QASFTTSMEW 1.500 676 27 165 SSDKMVCYLL 1.500 677 28 111NNVVLEAPFL 1.500 678 29 196 IAELKEKIVL 1.350 679 30 26 RAIDQASFTT 1.200680 31 207 HNRLKSLMKI 1.200 681 32 204 VLTHNRLKSL 1.000 682 33 137CGSCGIPVGF 1.000 683 34 86 VLADSVHLAW 1.000 684 35 41 TQVVKGSSPL 1.000685 36 139 SCGIPVGFHL 1.000 686 37 92 HLAWDLSRSL 1.000 687 38 69WLQPERCAVF 1.000 688 39 78 FQCAQCHAVL 1.000 689 40 110 TNNVVLEAPF 1.000690 41 195 KIAELKEKIV 0.800 691 42 209 RLKSLMKILS 0.600 692 43 15PPRGDFCGGT 0.600 693 44 71 QPERCAVFQC 0.600 694 45 89 DSVHLAWDLS 0.500695 46 24 TERAIDQASF 0.450 696 47 170 VCYLLKTKAI 0.400 697 48 178AIVNASEMDI 0.400 698 49 133 NLLFCGSCGI 0.400 699 50 187 IQNVPLSEKI 0.400700

Table XIX: Motifs and Post-Translational Modifications N-glycosylationsite 181-184 NASE (SEQ ID NO: 735)

Protein kinase C phosphorylation site

Number of matches: 4

-   -   1 24-26 TER    -   2 126-128 SLK    -   3 166-168 SDK    -   4 193-195 SEK

Casein kinase II phosphorylation site

Number of matches: 3 1  35-38 TSME (SEQ ID NO: 736) 2 183-186 SEMD (SEQID NO: 737) 3 225-228 SKPE (SEQ ID NO: 738)

N-myristoylation site

Number of matches: 5 1  23-28 GTERAI (SEQ ID NO: 739) 2 122-127 GIEGSL(SEQ ID NO: 740) 3 125-130 GSLKGS (SEQ ID NO: 741) 4 129-134 GSTYNL (SEQID NO: 742) 5 141-146 GIPVGF (SEQ ID NO: 743)

RGD Cell attachment sequence

-   -   17-19 RGD

Cytochrome c family heme-binding site signature 80-85 CAQCHA (SEQ ID NO:744)

TABLE XX Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome b N 68% Cytochrome b(N- membrane bound oxidase,terminal)/b6/petB generate superoxide ig 19% Immunoglobulin domains areone hundred amino acids domain long and include a conserved intradomaindisulfide bond. WD40 18% WD domain, G-beta tandem repeats of about 40residues, repeat each containing a Trp-Asp motif. Function in signaltransduction and protein interaction PDZ 23% PDZ domain may function intargeting signaling molecules to sub-membranous sites LRR 28% LeucineRich short sequence motifs involved in Repeat protein-proteininteractions pkinase 23% Protein kinase conserved catalytic core commonto both domain serine/threonine and tyrosine protein kinases containingan ATP binding site and a catalytic site PH 16% PH domain pleckstrinhomology involved in intra- cellular signaling or as constituents of thecytoskeleton EGF 34% EGF-like 30-40 amino-acid long found in the domainextracellular domain of membrane-bound proteins or in secreted proteinsrvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ankrepeat Cytoplasmic protein, associates integral membrane proteins to thecytoskeleton oxidored q1 32% NADH-Ubiquinone/ membrane associated.Involved in proton plastoquinone (complex translocation across themembrane I), various chains efhand 24% EF hand calcium-binding domain,consists of a12 residue loop flanked on both sides by a 12 residuealpha-helical domain rvp 79% Retroviral aspartyl Aspartyl or acidproteases, centered protease on a catalytic aspartyl residue Collagen42% Collagen triple extracellular structural proteins involved helixrepeat in formation of connective tissue. The (20 copies) sequenceconsists of the G-X-Y and the polypeptide chains forms a triple helix.fn3 20% Fibronectin Located in the extracellular ligand- type IIIbinding region of receptors and is about domain 200 amino acid residueslong with two pairs of cysteines involved in disulfide bonds 7tm 1 19% 7transmembrane seven hydrophobic transmembrane regions, receptor(rhodopsin with the N-terminus located extracellularly family) while theC-terminus is cytoplasmic. Signal through G proteins

TABLE XXV Protein Properties Bioinformatic Program Outcome ORF ORFFinder 13-702 (includes stop) Protein Length 229 amino acidsTransmembrane TM Pred one TM at aa 129-149 region HMMTop one TM at aa134-158 Sosui indicates no TM, soluble protein TMHMM indicates no TMSignal Peptide Signal P indicates no signal pI pI/MW tool pI 7.02Molecular weight pI/MW tool 24.69 kDa Localization PSORT Cytoplasmic 65%Mitochondrial 10% PSORT II Mitochondrial 60.9% Cytoplamic 21.7% MotifsPfam no motif detected Prints no significant motif Blocks Soybeantrypsin inhibitor protease family, Cytochrome c Prosite Cytochrome cfamily, heme binding signature

1. An isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ NO:
 728. 2. The isolated nucleic acid of claim 1, wherein the nucleotide sequence is that shown in SEQ ID NO:
 727. 3. The isolated nucleic acid of claim 2, wherein the nucleotide sequence is that from nucleotide residue number 13 through nucleotide residue number
 699. 4. A recombinant expression vector that comprising the nucleotide sequence of claim
 3. 5. The recombinant expression vector of claim 4, wherein the vector is replication competent in a mammalian host.
 6. A host cell comprising the expression vector of claim
 4. 7. The host cell of claim 6, wherein the cell is a prokaryotic cell.
 8. The host cell of claim 6, wherein the cell is a eucaryotic cell.
 9. A viral vector comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO:728, wherein the nucleotide sequence is operably linked to promoter for expression of SEQ ID NO: 728 in a mammalian cell.
 10. The viral vector of claim 9, were in the nucleotide sequence is SEQ ID NO:
 727. 11. The viral vector of claim 9, further comprising a pharmaceutically acceptable carrier.
 12. The viral vector of claim 9, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to SEQ ID NO:
 728. 13. The viral vector of claim 9, wherein the isolated nucleic acid molecule which encodes SEQ ID NO: 728 is SEQ ID NO:
 727. 14. The viral vector of claim 9, wherein the promoter is a viral promoter.
 15. The viral vector of claim 9, wherein the viral promoter is cytomegalovirus promoter (CMV).
 16. A method of generating a mammalian immune response directed to a protein, comprising: exposing cells of the mammal's immune system to an immunogenic portion of SEQ ID NO:
 728. 17. The method of claim 16, wherein the generated immune response comprises an activation of a T cell or a B cell in the mammal.
 18. The method of claim 17, wherein the immune response comprises an activated B cell that generates antibodies that bind specifically to the SEQ ID NO:
 728. 19. The method of claim 17, wherein the immunogenic portion of SEQ ID NO: 728 is provided by a viral expression vector.
 20. The method of claim 19, wherein the viral expression vector is a viral vector comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO:728, wherein the nucleotide sequence is operably linked to promoter for expression of SEQ ID NO: 728 in a mammalian cell. 